# Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept

Arnaud Paré, Baptiste Charbonnier, Joëlle Veziers, Caroline Vignes, Maeva Dutilleul, Gonzague de Pinieux, Boris Laure, Adeline Bossard, Annaëlle Saucet-Zerbib, Gwenola Touzot-Jourde, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Arnaud Paré, Baptiste Charbonnier, Joëlle Veziers, Caroline Vignes, Maeva Dutilleul, et al. Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept. Acta Biomaterialia, 2022, 154, pp.626-640. 10.1016/j.actbio.2022.09.071. hal-04592054

HAL Id: hal-04592054

https://nantes-universite.hal.science/hal-04592054

Submitted on 29 May 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept

Arnaud Paré, Baptiste Charbonnier, Joëlle Veziers, Caroline Vignes, Maeva

Dutilleul, Gonzague de Pinieux, Boris Laure, Adeline Bossard, Annaëlle

Saucet-Zerbib, Gwenola Touzot-Jourde, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Arnaud Paré, Baptiste Charbonnier, Joëlle Veziers, Caroline Vignes, Maeva Dutilleul, et al. Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept. Acta Biomaterialia, 2022, 154, pp.626-640. 10.1016/j.actbio.2022.09.071. hal-04592054

HAL Id: hal-04592054

https://nantes-universite.hal.science/hal-04592054v1

Submitted on 29 May 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

ELSEVIER

Contents lists available at ScienceDirect

Acta Biomaterialia

journal homepage: www.elsevier.com/locate/actbio

<figure><img src="image_4.png" /></figure>

Full length article

# Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept

<figure><img src="image_5.png" /></figure>

Arnaud Paré<sup>a,b</sup>, Baptiste Charbonnier<sup>a,c,f</sup>, Joëlle Veziers<sup>a</sup>, Caroline Vignes<sup>a</sup>, Maeva Dutilleul<sup>a</sup>, Gonzague De Pinieux<sup>d</sup>, Boris Laure<sup>b</sup>, Adeline Bossard<sup>e</sup>, Annaëlle Saucet-Zerbib<sup>e</sup>, Gwenola Touzot-Jourde<sup>a,e</sup>, Pierre Weiss<sup>a</sup>, Pierre Corre<sup>a,f</sup>, Olivier Gauthier<sup>a,e</sup>, David Marchat<sup>c,*</sup>

ARTICLE INFO

Article history:
Received 20 April 2022
Revised 9 September 2022
Accepted 28 September 2022
Available online 6 October 2022

Keywords:
Calcium phosphates
Additive manufacturing
Segmental mandibulectomy
Bone regeneration
Surgical model

ABSTRACT

The reconstruction of massive segmental mandibular bone defects (SMDs) remains challenging even today; the current gold standard in human clinics being vascularized bone transplantation (VBT). As alternative to this onerous approach, bone tissue engineering strategies have been widely investigated. However, they displayed limited clinical success, particularly in failing to address the essential problem of quick vascularization of the implant. Although routinely used in clinics, the insertion of intrinsic vascularization in bioengineered constructs for the rapid formation of a feeding angiosome remains uncommon.

In a clinically relevant model (sheep), a custom calcium phosphate-based bioceramic soaked with autologous bone marrow and perfused by an arteriovenous loop was tested to regenerate a massive SMD and was compared to VBT (clinical standard). Animals did not support well the VBT treatment, and the study was aborted 2 weeks after surgery due to ethical and animal welfare considerations. SMD regeneration was successful with the custom vascularized bone construct. Implants were well osseointegrated and vascularized after only 3 months of implantation and totally entrapped in lamellar bone after 12 months; a healthy yellow bone marrow filled the remaining space.

Statement of significance

Regenerative medicine struggles with the generation of large functional bone volume. Among them segmental mandibular defects are particularly challenging to restore. The standard of care, based on bone free flaps, still displays ethical and technical drawbacks (e.g., donor site morbidity).

Modern engineering technologies (e.g., 3D printing, digital chain) were combined to relevant surgical techniques to provide a pre-clinical proof of concept, investigating for the benefits of such a strategy in bone-related regenerative field.

Results proved that a synthetic-biologics-free approach is able to regenerate a critical size segmental mandibular defect of 15 cm³ in a relevant preclinical model, mimicking real life scenarii of segmental mandibular defect, with a full physiological regeneration of the defect after 12 months.

© 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc.

This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/))

<footnote><sup>a</sup>INSERM, U 1229, Laboratory of Regenerative Medicine and Skeleton, RMeS, Nantes Université, 1 Place Alexis Ricordeau, Nantes 44042, France</footnote>

<footnote><sup>b</sup>Department of Maxillofacial and Plastic surgery, Burn Unit, University Hospital of Tours, Trousseau Hospital, Avenue de la République, Chambray lès Tours 37170, France</footnote>

<footnote><sup>c</sup>Mines Saint-Étienne, Univ Jean Monnet, INSERM, U 1059 Sainbiose, 42023, Saint-Étienne, France</footnote>

<footnote><sup>d</sup>Department of Pathology, University Hospital of Tours, Trousseau Hospital, Avenue de la République, Chambray lès Tours 37170, France</footnote>

<footnote><sup>e</sup>ONIRIS Nantes-Atlantic College of Veterinary Medicine, Research Center of Preclinical Invesitagtion (CRIP), Site de la Chantrerie, 101 route de Gachet, Nantes 44307, France</footnote>

<footnote><sup>f</sup>Clinique de Stomatologie et Chirurgie Maxillo-Faciale, Nantes University Hospital, 1 Place Alexis Ricordeau, Nantes 44042, France</footnote>

<footnote>* Corresponding author.
E-mail address: marchat@emse.fr (D. Marchat).</footnote>

<footnote n="1">¹ Co-first author.</footnote>
<figure><img src="image_3.png" /></figure>

ELSEVIER

Contents lists available at [ScienceDirect](https://www.sciencedirect.com)

Acta Biomaterialia

journal homepage: [www.elsevier.com/locate/actbio](https://www.elsevier.com/locate/actbio)

<figure><img src="image_4.png" /></figure>

Full length article

# Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept

<figure><img src="image_5.png" /></figure>

Arnaud Paré<sup>a,b</sup>, Baptiste Charbonnier<sup>a,c,f</sup>, Joëlle Veziers<sup>a</sup>, Caroline Vignes<sup>a</sup>, Maeva Dutilleul<sup>a</sup>, Gonzague De Pinieux<sup>d</sup>, Boris Laure<sup>b</sup>, Adeline Bossard<sup>e</sup>, Annaëlle Saucet-Zerbib<sup>e</sup>, Gwenola Touzot-Jourde<sup>a,e</sup>, Pierre Weiss<sup>a</sup>, Pierre Corre<sup>a,f</sup>, Olivier Gauthier<sup>a,e</sup>, David Marchat<sup>c,*</sup>

ARTICLE INFO

Article history:
Received 20 April 2022
Revised 9 September 2022
Accepted 28 September 2022
Available online 6 October 2022

Keywords:
Calcium phosphates
Additive manufacturing
Segmental mandibulectomy
Bone regeneration
Surgical model

ABSTRACT

The reconstruction of massive segmental mandibular bone defects (SMDs) remains challenging even today; the current gold standard in human clinics being vascularized bone transplantation (VBT). As alternative to this onerous approach, bone tissue engineering strategies have been widely investigated. However, they displayed limited clinical success, particularly in failing to address the essential problem of quick vascularization of the implant. Although routinely used in clinics, the insertion of intrinsic vascularization in bioengineered constructs for the rapid formation of a feeding angiosome remains uncommon.

In a clinically relevant model (sheep), a custom calcium phosphate-based bioceramic soaked with autologous bone marrow and perfused by an arteriovenous loop was tested to regenerate a massive SMD and was compared to VBT (clinical standard). Animals did not support well the VBT treatment, and the study was aborted 2 weeks after surgery due to ethical and animal welfare considerations. SMD regeneration was successful with the custom vascularized bone construct. Implants were well osseointegrated and vascularized after only 3 months of implantation and totally entrapped in lamellar bone after 12 months; a healthy yellow bone marrow filled the remaining space.

Statement of significance

Regenerative medicine struggles with the generation of large functional bone volume. Among them segmental mandibular defects are particularly challenging to restore. The standard of care, based on bone free flaps, still displays ethical and technical drawbacks (e.g., donor site morbidity).

Modern engineering technologies (e.g., 3D printing, digital chain) were combined to relevant surgical techniques to provide a pre-clinical proof of concept, investigating for the benefits of such a strategy in bone-related regenerative field.

Results proved that a synthetic-biologics-free approach is able to regenerate a critical size segmental mandibular defect of 15 cm³ in a relevant preclinical model, mimicking real life scenarii of segmental mandibular defect, with a full physiological regeneration of the defect after 12 months.

© 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc.
This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/))

<footnote><sup>a</sup>INSERM, U 1229, Laboratory of Regenerative Medicine and Skeleton, RMeS, Nantes Université, 1 Place Alexis Ricordeau, Nantes 44042, France</footnote>

<footnote><sup>b</sup>Department of Maxillofacial and Plastic surgery, Burn Unit, University Hospital of Tours, Trousseau Hospital, Avenue de la République, Chambray lès Tours 37170, France</footnote>

<footnote><sup>c</sup>Mines Saint-Étienne, Univ Jean Monnet, INSERM, U 1059 Sainbiose, 42023, Saint-Étienne, France</footnote>

<footnote><sup>d</sup>Department of Pathology, University Hospital of Tours, Trousseau Hospital, Avenue de la République, Chambray lès Tours 37170, France</footnote>

<footnote><sup>e</sup>ONIRIS Nantes-Atlantic College of Veterinary Medicine, Research Center of Preclinical Invesitagtion (CRIP), Site de la Chantrerie, 101 route de Gachet, Nantes 44307, France</footnote>

<footnote><sup>f</sup>Clinique de Stomatologie et Chirurgie Maxillo-Faciale, Nantes University Hospital, 1 Place Alexis Ricordeau, Nantes 44042, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: marchat@emse.fr (D. Marchat).</footnote>

<footnote n="1">¹ Co-first author.</footnote>
# 1. Introduction

Currently, vascularized bone transplants (VBTs) are the gold standard in human clinics for the regeneration of large segmental mandibular defects (SMDs), offering satisfactory functional restoration (e.g., mastication, swallowing, breathing and phonation), especially when the surrounding tissues have been compromised (e.g., poor soft tissue coverage, irradiation, infection). Indeed, in these particular settings, mandibular reconstruction with non-vascularized autologous bone grafts or alloplastic implants have displayed a high failure rate [1-5]. Moreover, recent technical improvements (e.g., harvest refinements, computer-assisted surgery) have clearly optimized the reconstruction procedures (e.g., morphological outcomes, operative time) [6-8]. However, the disadvantages of VBT remain, such as the harvesting of healthy bone from patients already affected by a severe medical condition [9], the limited tissue availability, and the anatomical mismatch between donor and recipient sites that often hinder the restoration of normal function [10-12].

Alternative strategies to VBT have been extensively investigated for the past 30 years. Cell therapies and bone tissue engineering (BTE) approaches based on the combination of expanded patient cells (e.g., mesenchymal stem cells, MSCs), synthetic bioactive substances (e.g., bone morphogenetic proteins, teriparatide) and/or natural or synthetic biomaterials (e.g., collagen sponge, coral, calcium phosphates: CaP, polycaprolactone) have demonstrated promising potential for the regeneration of craniofacial defects [13-19]. However, the translation of these approaches to clinical settings remains complex due to challenges in large-scale expansion of MSCs, potential side effects of synthetic biology approaches, and on the whole, the lack of an evident risk/benefits ratio for mandibular reconstruction [18,20-25]. A more relevant and safer alternative, already applied in clinics, is the extemporaneous combination of synthetic CaP-based biomaterials with autologous bone marrow (BM) as a natural source of various angiogenic and osteogenic cells and factors (e.g., mesenchymal and hematopoietic progenitor cells) fostering bone formation [26]. As such, reconstruction of craniofacial defects has been routinely performed in clinics in a single-step intervention using CaP-based biomaterials soaked with fresh total BM aspirate (without further cell isolation or expansion) [27-31].

For massive defects or compromised environments [32], the issue is not so much the nature of the bioactive substances and cells used to enhance the biomaterial regenerative potential but the way to keep them alive and functional to directly or indirectly (i.e., paracrine) stimulate the formation of new bone tissue [33-38]. Indeed, the extrinsic vascularization, i.e., the small blood vessels and capillaries that quickly invade the periphery of the scaffold (≈ 1-3 mm) [39,40], are unfortunately not sufficient to provide oxygen and nutrients and remove waste up to the core of the implant. Consequently, only limited or 2D sheet-shaped tissue engineered volumes can be sustained. In contrast, the diversion of intrinsic vasculature (e.g., arteriovenous bundle AVB or loop AVL) within the scaffold has allowed the formation of large bone volumes and/or the regeneration of critical-sized segmental bone defects [41-43] due to the rapid surgically induced development of a functional angiosome [44,45].

Extensively studied in preclinical setups in both small and large animal models [39,32,46-48], the combination of AVL with bone filler and/or active substances is nonetheless rather unusual in clinics for the regeneration of SMD. The complexity of the microsurgical procedure but mainly the lack of suitable biomaterials to support tissue growth are at stake. Standard bone fillers, such as biphasic calcium phosphate (BCP)-based granules or porous blocks, are not designed and poorly adapted for such treatment and de-

manding environments (e.g., limited implant-defect matching, unsuitable mechanical properties, micromotions between blocks or granules, migration out of the defect).

Based on those observations, the proposed study was designed on 3 main aspects to fulfill the current needs for the regeneration of massive segmental mandibular bone defects: (i) the tailored design and production of BCP-based implants specifically designed for the surgical strategy and the challenging SMD environment, (ii) the addition of bone marrow to enhance angiogenesis and osteogenesis within the defect without the drawbacks of processed cells and synthetic active substances, and (iii) the presence of an intrinsic vascularization (AVL) to provide a favorable biological environment for the development of an angiosome that will sustain bone formation.

This work presents a proof of concept of a bottom-up biomaterial- and surgical-based strategy to regenerate massive SMD in sheep, without recourse to any synthetic biologics and expanded cells for a faster translation to clinics. As there is no positive control for such an animal model [49], regeneration of the SMD was also been assessed with a vascularized bone transplant (VBT), which is the reference treatment in human practice.

# 2. Materials & methods

## 2.1. *Manufacturing of the tailored BCP implant*

Implants were designed from a segmental mandibular bone defect virtually created using ScanIP software (Simpleware, UK) and a scanner dataset of a skull sheep exported in DICOM format (Fig. S1A in the Supporting Information). The global architecture was thought to allow perfect contact between itself and the adjacent host tissues according to 3D reconstructed CT images from a representative sheep of the batch, to accommodate an arteriovenous fistula, to avoid injuring the animal with angular parts and to facilitate the work of the surgeon (e.g., holes to fix sutures (Fig. S1B in the Supporting Information). The macroporous architecture of the implants (e.g., curvature of the ceramic surface, macropore size) was designed to promote osteogenesis and blood vessel growth by using a gyroid internal structure [40,50,51] with a pore volume fraction of 40% wherein a sphere of 800 µm could freely move (Fig. S1C in the Supporting Information); additional information is available in Paré et al.'s manuscript [52].

BCP implants were produced through the impregnation of 3D-printed wax molds by a calcium phosphate suspension, as detailed in Charbonnier et al. [53]. Briefly, molds designed as the negative structure of the intended implant, with ScanIP software (Simpleware, UK), were built layer-by-layer (50 µm thickness) with a drop-on-demand 3D printer (3Z Max, Solidscape, Multistation, Dinard). To obtain the analogous implants after sintering, the dimensions of the molds and the parameters of the gyroid structure were adjusted considering an isotropic shrinkage of 9.0%. (Fig. S1D in the Supporting Information).

Once printed and cleaned, the molds were impregnated with a BCP ceramic powder suspension (i.e., slurry). After drying overnight at room temperature, green bodies were cleaned of all excess dried slurry using a surgical blade (Swann-Morton, UK); Fig. S1E (Supporting Information) shows the green materials after this step.

Then, cleaned green bodies were heat-treated in a debinding furnace (Carbolite, UK) up to 500°C to remove the organic substances, and finally sintered at 1100°C for 2 h under air (ramps 4°C min⁻¹, Nabertherm LH40/14, Germany).

The BCP slurry was prepared by blending 42.8% (w/w) hydroxyapatite powder (HA, Ca₁₀(PO₄)₆(OH)₂), 28.6% w/w β-tricalcium phosphate powder (β-TCP, Ca₃(PO₄)₂), 27.7% w/w pure water, and
# 1. Introduction

Currently, vascularized bone transplants (VBTs) are the gold standard in human clinics for the regeneration of large segmental mandibular defects (SMDs), offering satisfactory functional restoration (e.g., mastication, swallowing, breathing and phonation), especially when the surrounding tissues have been compromised (e.g., poor soft tissue coverage, irradiation, infection). Indeed, in these particular settings, mandibular reconstruction with non-vascularized autologous bone grafts or alloplastic implants have displayed a high failure rate [1-5]. Moreover, recent technical improvements (e.g., harvest refinements, computer-assisted surgery) have clearly optimized the reconstruction procedures (e.g., morphological outcomes, operative time) [6-8]. However, the disadvantages of VBT remain, such as the harvesting of healthy bone from patients already affected by a severe medical condition [9], the limited tissue availability, and the anatomical mismatch between donor and recipient sites that often hinder the restoration of normal function [10-12].

Alternative strategies to VBT have been extensively investigated for the past 30 years. Cell therapies and bone tissue engineering (BTE) approaches based on the combination of expanded patient cells (e.g., mesenchymal stem cells, MSCs), synthetic bioactive substances (e.g., bone morphogenetic proteins, teriparatide) and/or natural or synthetic biomaterials (e.g., collagen sponge, coral, calcium phosphates: CaP, polycaprolactone) have demonstrated promising potential for the regeneration of craniofacial defects [13-19]. However, the translation of these approaches to clinical settings remains complex due to challenges in large-scale expansion of MSCs, potential side effects of synthetic biology approaches, and on the whole, the lack of an evident risk/benefits ratio for mandibular reconstruction [18,20-25]. A more relevant and safer alternative, already applied in clinics, is the extemporaneous combination of synthetic CaP-based biomaterials with autologous bone marrow (BM) as a natural source of various angiogenic and osteogenic cells and factors (e.g., mesenchymal and hematopoietic progenitor cells) fostering bone formation [26]. As such, reconstruction of craniofacial defects has been routinely performed in clinics in a single-step intervention using CaP-based biomaterials soaked with fresh total BM aspirate (without further cell isolation or expansion) [27-31].

For massive defects or compromised environments [32], the issue is not so much the nature of the bioactive substances and cells used to enhance the biomaterial regenerative potential but the way to keep them alive and functional to directly or indirectly (i.e., paracrine) stimulate the formation of new bone tissue [33-38]. Indeed, the extrinsic vascularization, i.e., the small blood vessels and capillaries that quickly invade the periphery of the scaffold (≈ 1-3 mm) [39,40], are unfortunately not sufficient to provide oxygen and nutrients and remove waste up to the core of the implant. Consequently, only limited or 2D sheet-shaped tissue engineered volumes can be sustained. In contrast, the diversion of intrinsic vasculature (e.g., arteriovenous bundle AVB or loop AVL) within the scaffold has allowed the formation of large bone volumes and/or the regeneration of critical-sized segmental bone defects [41-43] due to the rapid surgically induced development of a functional angiosome [44,45].

Extensively studied in preclinical setups in both small and large animal models [39,32,46-48], the combination of AVL with bone filler and/or active substances is nonetheless rather unusual in clinics for the regeneration of SMD. The complexity of the microsurgical procedure but mainly the lack of suitable biomaterials to support tissue growth are at stake. Standard bone fillers, such as biphasic calcium phosphate (BCP)-based granules or porous blocks, are not designed and poorly adapted for such treatment and de-

manding environments (e.g., limited implant-defect matching, unsuitable mechanical properties, micromotions between blocks or granules, migration out of the defect).

Based on those observations, the proposed study was designed on 3 main aspects to fulfill the current needs for the regeneration of massive segmental mandibular bone defects: (i) the tailored design and production of BCP-based implants specifically designed for the surgical strategy and the challenging SMD environment, (ii) the addition of bone marrow to enhance angiogenesis and osteogenesis within the defect without the drawbacks of processed cells and synthetic active substances, and (iii) the presence of an intrinsic vascularization (AVL) to provide a favorable biological environment for the development of an angiosome that will sustain bone formation.

This work presents a proof of concept of a bottom-up biomaterial- and surgical-based strategy to regenerate massive SMD in sheep, without recourse to any synthetic biologics and expanded cells for a faster translation to clinics. As there is no positive control for such an animal model [49], regeneration of the SMD was also been assessed with a vascularized bone transplant (VBT), which is the reference treatment in human practice.

# 2. Materials & methods

## 2.1. *Manufacturing of the tailored BCP implant*

Implants were designed from a segmental mandibular bone defect virtually created using ScanIP software (Simpleware, UK) and a scanner dataset of a skull sheep exported in DICOM format (Fig. S1A in the Supporting Information). The global architecture was thought to allow perfect contact between itself and the adjacent host tissues according to 3D reconstructed CT images from a representative sheep of the batch, to accommodate an arteriovenous fistula, to avoid injuring the animal with angular parts and to facilitate the work of the surgeon (e.g., holes to fix sutures (Fig. S1B in the Supporting Information). The macroporous architecture of the implants (e.g., curvature of the ceramic surface, macropore size) was designed to promote osteogenesis and blood vessel growth by using a gyroid internal structure [40,50,51] with a pore volume fraction of 40% wherein a sphere of 800 µm could freely move (Fig. S1C in the Supporting Information); additional information is available in Paré et al.'s manuscript [52].

BCP implants were produced through the impregnation of 3D-printed wax molds by a calcium phosphate suspension, as detailed in Charbonnier et al. [53]. Briefly, molds designed as the negative structure of the intended implant, with ScanIP software (Simpleware, UK), were built layer-by-layer (50 µm thickness) with a drop-on-demand 3D printer (3Z Max, Solidscape, Multistation, Dinard). To obtain the analogous implants after sintering, the dimensions of the molds and the parameters of the gyroid structure were adjusted considering an isotropic shrinkage of 9.0%. (Fig. S1D in the Supporting Information).

Once printed and cleaned, the molds were impregnated with a BCP ceramic powder suspension (i.e., slurry). After drying overnight at room temperature, green bodies were cleaned of all excess dried slurry using a surgical blade (Swann-Morton, UK); Fig. S1E (Supporting Information) shows the green materials after this step.

Then, cleaned green bodies were heat-treated in a debinding furnace (Carbolite, UK) up to 500°C to remove the organic substances, and finally sintered at 1100°C for 2 h under air (ramps 4°C min⁻¹, Nabertherm LH40/14, Germany).

The BCP slurry was prepared by blending 42.8% (w/w) hydroxyapatite powder (HA, Ca₁₀(PO₄)₆(OH)₂), 28.6% w/w β-tricalcium phosphate powder (β-TCP, Ca₃(PO₄)₂), 27.7% w/w pure water, and
0.9% w/w dispersing agent (Darvan C-N, R. T. Vanderbilt Company Inc., USA) for 12 min at 170 rpm in a zirconia jar with 5 and 10 mm diameter zirconia balls (PM400, Retsch, Germany). Before mold impregnation, an organic binder (1.9% w/w, Duramax B-1000, Rohmand Haas, France) was mixed with the slurries at 140 rpm for 15 min using a propeller stirrer. HA and β-TCP powders, used to produce this slurry, were prepared by a conventional aqueous precipitation method followed by heat treatment to adjust their physicochemical properties; the complete method is detailed in Paré et al. [52]. Their specific surface areas were adjusted between 4 and 5 m² g⁻¹, respectively.

To evaluate the capability of the manufacturing process, 3D-printed implants were compared to their theoretical CAD models. To do so, the implants were imaged three-dimensionally using an X-ray computed microtomography system (Nanotom 180, GE Sensing & Inspection Technologies Phoenix X-ray, USA) with a resolution of 25 µm. After segmentation and meshing of the 3D images using DragonFly (ORS, Canada), .stl files of the produced bioceramics were superimposed on their CAD models, and then their differences were assessed with CloudCompare freeware (https://www.danielgm.net/cc/). The reported deviations included not only the manufacturing process capability but also the possible inaccuracies and errors throughout the analysis process (e.g., µCT, superimposition).

## 2.2. Animals

Adult sheep were obtained from a certified breeding center (GAEC Heas, Ligné, France) and acclimatized for 2 weeks before the surgical procedure. All sheep (Vendean strain) were female aged 4 to 7 years with an average weight of 80 kg (75-84 kg). The study design originally included 15 sheep for the in vivo experiments: 6 for the experimental group (Sh1-6), 6 for the clinical control (Sh7-12) and 3 for the cadaveric study (Sh13-15). As this study was designed as a proof of concept to evaluate the feasibility of innovative approaches (e.g., positive control, reconstruction strategy), it was designed with a limited number of animals for ethical reasons: 5 animals per group (Sh1-5 and Sh7-11) for an implantation time of 3 months, and only one (Sh6 and Sh12) for an implantation time of 12 months. A tendency to be confirmed in a more extensive pre-clinical study rather than statistical data were consequently expected.

Animal study was conducted according to European directive 2010/63/EU and ARRIVE guideline [54]. The experimental design was approved prior to the animal experiments by the French Ministry of Research and Education (APAFIS 11192), by the Pays de la Loire Ethics Committee (France) and by the local ethic and welfare committee (F.44-271).

## 2.3. Cadaveric study

A cadaveric study was first performed to precisely define the segmental mandible defect (e.g., size, location) and to gather realistic information about the vascular anatomy of the animals. In short, the most suitable vascular axis for preforming an AVL and predicting its path through the custom-made implant was investigated. Six dissections were performed on 3 fresh cadaveric adult sheep (left and right sides). A cervical approach was carried out to limit oral contamination of the surgical site. A segmental defect of ≈35 mm long and ≈55 mm high which involved the angle of the mandible (Figs. S1A and S2A in the Supporting Information) was generated, as it is a common location of odontogenic tumors (e.g., ameloblastoma, ossifying fibroma) [55,56] and to avoid tooth removal or mucosal wounds that could impede postoperative feeding and functional outcomes. A submandibular incision offered optimal exposure and easy dissection of the facial vein (FV, Fig. S2B in the

Supporting Information). Due to the absence of the facial artery in the sheep model, the best vascular axis was the transverse facial artery (TFA). The harvesting of a vascularized bone transplant from the iliac crest (clinical control) was also investigated as well as its vascularization by the deep circumflex iliac pedicle (DCIP, Fig. S2C in the Supporting Information). The extremity of the FV had an average length of 12 cm with a diameter close to that of the TFA (3.5 and 2.5 mm, respectively). The average length of the TFA was 5 cm, allowing AVL creation, which was placed through the masseter muscle to maintain the AVL in the mandible defect.

## 2.4. Surgery

### 2.4.1. Anesthesia

General anesthesia was administered using an intrajugular catheter (16 gauges). Premedication with diazepam (0.25 mg kg⁻¹, ALIUM ROCHE NDH, 5 mg mL⁻¹, Roche, Bâle, Switzerland) and morphine (0.5 mg kg⁻¹, MORPHINE COOPER, 10 mg mL⁻¹, Cooper, Melun, France) was first administered. Then, the animal was induced with ketamine (3 to 5 mg kg⁻¹, IMALGENE 1000D NDV, Merial, Lyon, France) and propofol (5 to 6 mg kg⁻¹, PROPOVET MULTIDOSE NDV, 10 mg mL⁻¹, Zoetis, Madison, USA) followed by endotracheal intubation. During the procedure, maintenance of general anesthesia was ensured by sevoflurane inhalational anesthetic (SEVOFLO 100%, ABBOTT, Chicago, USA) in 100% oxygen, and normal saline (10 mL kg⁻¹ h⁻¹, RINGER LACTATE NDV, BRAUN, Melsungen, Germany) was perfused. Intravenous prophylactic antibiotic therapy was given pre- and perioperatively with amoxicillin (20 mg kg⁻¹ 2 h⁻¹, CLAMOXYL NDV, ZoetisMadison, USA).

### 2.4.2. Bone marrow harvesting

Total bone marrow (TBM) was systematically harvested under general anesthesia during the surgical procedure at the ipsilateral side of the mandible repair. A 2 cm longitudinal incision was made in front of the dorsal iliac crest, and bone was exposed. A 2 mm hole was drilled, and TBM (5 mL) was aspirated from the iliac crest using an 11-gauge Jamshidi needle (COVETO, La Guyonnière, France). The donor site was closed using absorbable sutures for the subcutaneous and dermal layers (Monocryl 2.0, Ethicon, Somerville, USA) and the skin (Vicryl 3.0, Ethicon).

### 2.4.3. Surgical procedure

In a right lateral decubitus position with the head extended, the mandibular region was shaved and disinfected using chlorhexidine-ethanol (chlorhexidine alcohol 0.5%, Gilbert, Herouville-Saint-Pierre, France). A submandibular incision of 10 cm was made with preservation of the facial vein. The periosteum covering the angle of the mandible was detached from the bone and raised with the masseter muscle. Analogous dissection of the medial pterygoid muscle was also carried out to expose the inner part of the mandible. Before mandibulectomy, the osteosynthesis plate (2.7 mm, Stryker France, Lyon, France) was shaped, and fixation holes were drilled in the mandible to avoid postoperative malocclusion. A segmental defect of the mandible angle of ≈15 cm³ was performed (≈35 mm long and ≈55 mm high, Fig. S3A in the Supporting Information), using a standardized implant-shaped template as a surgical guide (3D printed polylactic acid tool), and then generated using an oscillating saw (Stryker). Extra care was provided to preserve as much of the inferior alveolar pedicle as possible during the osteotomy – the latter was ligated before the complete removal of the bone. The SMD was then stabilized by fixing the pre-shaped rigid plate with 3 ten-millimeter screws in the holes previously created on the rostral and caudal sides of the mandible. The FV and the transverse facial artery (TFA) were then dissected in view of the vascular anastomoses.
0.9% w/w dispersing agent (Darvan C-N, R. T. Vanderbilt Company Inc., USA) for 12 min at 170 rpm in a zirconia jar with 5 and 10 mm diameter zirconia balls (PM400, Retsch, Germany). Before mold impregnation, an organic binder (1.9% w/w, Duramax B-1000, Rohmand Haas, France) was mixed with the slurries at 140 rpm for 15 min using a propeller stirrer. HA and β-TCP powders, used to produce this slurry, were prepared by a conventional aqueous precipitation method followed by heat treatment to adjust their physicochemical properties; the complete method is detailed in Paré et al. [52]. Their specific surface areas were adjusted between 4 and 5 m² g⁻¹, respectively.

To evaluate the capability of the manufacturing process, 3D-printed implants were compared to their theoretical CAD models. To do so, the implants were imaged three-dimensionally using an X-ray computed microtomography system (Nanotom 180, GE Sensing & Inspection Technologies Phoenix X-ray, USA) with a resolution of 25 µm. After segmentation and meshing of the 3D images using DragonFly (ORS, Canada), .stl files of the produced bioceramics were superimposed on their CAD models, and then their differences were assessed with CloudCompare freeware (https://www.danielgm.net/cc/). The reported deviations included not only the manufacturing process capability but also the possible inaccuracies and errors throughout the analysis process (e.g., µCT, superimposition).

## 2.2. Animals

Adult sheep were obtained from a certified breeding center (GAEC Heas, Ligné, France) and acclimatized for 2 weeks before the surgical procedure. All sheep (Vendean strain) were female aged 4 to 7 years with an average weight of 80 kg (75-84 kg). The study design originally included 15 sheep for the in vivo experiments: 6 for the experimental group (Sh1-6), 6 for the clinical control (Sh7-12) and 3 for the cadaveric study (Sh13-15). As this study was designed as a proof of concept to evaluate the feasibility of innovative approaches (e.g., positive control, reconstruction strategy), it was designed with a limited number of animals for ethical reasons: 5 animals per group (Sh1-5 and Sh7-11) for an implantation time of 3 months, and only one (Sh6 and Sh12) for an implantation time of 12 months. A tendency to be confirmed in a more extensive pre-clinical study rather than statistical data were consequently expected.

Animal study was conducted according to European directive 2010/63/EU and ARRIVE guideline [54]. The experimental design was approved prior to the animal experiments by the French Ministry of Research and Education (APAFIS 11192), by the Pays de la Loire Ethics Committee (France) and by the local ethic and welfare committee (F.44-271).

## 2.3. Cadaveric study

A cadaveric study was first performed to precisely define the segmental mandible defect (e.g., size, location) and to gather realistic information about the vascular anatomy of the animals. In short, the most suitable vascular axis for preforming an AVL and predicting its path through the custom-made implant was investigated. Six dissections were performed on 3 fresh cadaveric adult sheep (left and right sides). A cervical approach was carried out to limit oral contamination of the surgical site. A segmental defect of ≈35 mm long and ≈55 mm high which involved the angle of the mandible (Figs. S1A and S2A in the Supporting Information) was generated, as it is a common location of odontogenic tumors (e.g., ameloblastoma, ossifying fibroma) [55,56] and to avoid tooth removal or mucosal wounds that could impede postoperative feeding and functional outcomes. A submandibular incision offered optimal exposure and easy dissection of the facial vein (FV, Fig. S2B in the

Supporting Information). Due to the absence of the facial artery in the sheep model, the best vascular axis was the transverse facial artery (TFA). The harvesting of a vascularized bone transplant from the iliac crest (clinical control) was also investigated as well as its vascularization by the deep circumflex iliac pedicle (DCIP, Fig. S2C in the Supporting Information). The extremity of the FV had an average length of 12 cm with a diameter close to that of the TFA (3.5 and 2.5 mm, respectively). The average length of the TFA was 5 cm, allowing AVL creation, which was placed through the masseter muscle to maintain the AVL in the mandible defect.

## 2.4. Surgery

### 2.4.1. Anesthesia

General anesthesia was administered using an intrajugular catheter (16 gauges). Premedication with diazepam (0.25 mg kg⁻¹, ALIUM ROCHE NDH, 5 mg mL⁻¹, Roche, Bâle, Switzerland) and morphine (0.5 mg kg⁻¹, MORPHINE COOPER, 10 mg mL⁻¹, Cooper, Melun, France) was first administered. Then, the animal was induced with ketamine (3 to 5 mg kg⁻¹, IMALGENE 1000D NDV, Merial, Lyon, France) and propofol (5 to 6 mg kg⁻¹, PROPOVET MULTIDOSE NDV, 10 mg mL⁻¹, Zoetis, Madison, USA) followed by endotracheal intubation. During the procedure, maintenance of general anesthesia was ensured by sevoflurane inhalational anesthetic (SEVOFLO 100%, ABBOTT, Chicago, USA) in 100% oxygen, and normal saline (10 mL kg⁻¹ h⁻¹, RINGER LACTATE NDV, BRAUN, Melsungen, Germany) was perfused. Intravenous prophylactic antibiotic therapy was given pre- and perioperatively with amoxicillin (20 mg kg⁻¹ 2 h⁻¹, CLAMOXYL NDV, ZoetisMadison, USA).

### 2.4.2. Bone marrow harvesting

Total bone marrow (TBM) was systematically harvested under general anesthesia during the surgical procedure at the ipsilateral side of the mandible repair. A 2 cm longitudinal incision was made in front of the dorsal iliac crest, and bone was exposed. A 2 mm hole was drilled, and TBM (5 mL) was aspirated from the iliac crest using an 11-gauge Jamshidi needle (COVETO, La Guyonnière, France). The donor site was closed using absorbable sutures for the subcutaneous and dermal layers (Monocryl 2.0, Ethicon, Somerville, USA) and the skin (Vicryl 3.0, Ethicon).

### 2.4.3. Surgical procedure

In a right lateral decubitus position with the head extended, the mandibular region was shaved and disinfected using chlorhexidine-ethanol (chlorhexidine alcohol 0.5%, Gilbert, Herouville-Saint-Pierre, France). A submandibular incision of 10 cm was made with preservation of the facial vein. The periosteum covering the angle of the mandible was detached from the bone and raised with the masseter muscle. Analogous dissection of the medial pterygoid muscle was also carried out to expose the inner part of the mandible. Before mandibulectomy, the osteosynthesis plate (2.7 mm, Stryker France, Lyon, France) was shaped, and fixation holes were drilled in the mandible to avoid postoperative malocclusion. A segmental defect of the mandible angle of ≈15 cm³ was performed (≈35 mm long and ≈55 mm high, Fig. S3A in the Supporting Information), using a standardized implant-shaped template as a surgical guide (3D printed polylactic acid tool), and then generated using an oscillating saw (Stryker). Extra care was provided to preserve as much of the inferior alveolar pedicle as possible during the osteotomy – the latter was ligated before the complete removal of the bone. The SMD was then stabilized by fixing the pre-shaped rigid plate with 3 ten-millimeter screws in the holes previously created on the rostral and caudal sides of the mandible. The FV and the transverse facial artery (TFA) were then dissected in view of the vascular anastomoses.
For the experimental group (Sh1-Sh6), the BCP-based implant was first loaded with 5 mL of TBM (Fig. S3B in the Supporting Information) as described in Section 4.5.2. and placed into the SMD. A second plate (1.5 mm, Stryker France, Lyon, France) fixed at the basal edge of the scaffold using 2 ten-millimeter screws (Fig. S3C in the Supporting Information) was used to limit eventual motions of the implant under loading (e.g., mastication). Three minutes after an intravenous injection of heparin (50 UI kg⁻¹, HEPARINE CHOAY NDH, Sanofi-Aventis, Paris, France), the FTA and the FV were anastomosed under a microsurgical magnifier (8.0 polypropylene, Prolene, Ethicon) to create the AVL, which was diverted in the implant gutter (Fig. S3D in the Supporting Information).

For the clinical control group (Sh7-Sh12), reconstruction of the SMD was performed with an osseous vascularized free flap. In short, the iliac crest (ipsilateral side of the SMD) was exposed after incision of the skin and gentle dissection of the muscles (i.e., gluteus medius, tensor fascia latae and rectus abdominis). The DCIP was dissected up to the common iliac pedicle. Osteotomy of the iliac crest was then performed to obtain a vascularized bone flap matching the mandible defect (≈35 mm long x ≈55 mm high, Fig. S2C in the Supporting Information). Three minutes after an intravenous injection of heparin (50 UI kg⁻¹), the bone flap was harvested (section of the DCIP) and then placed in the SMD and fixed (osteosynthesis plate and 12 mm screws). The DCIP vein and artery were anastomosed to the FV and TFA (8.0 polypropylene), respectively.

A patency test for both the AVL and the free flap anastomoses was conducted before closure of the operative sites.

For both groups, the masseter and medial pterygoid muscles were sutured to the 2.7 mm-thick fixation plate using Vicryl 2/0 sutures. The wound was closed by bilayer suturing of the skin using Vicryl 3.0 (Etichon). The wound was cleaned with physiological serum and covered with a dry dressing. Meloxicam (0.5 mg kg⁻¹, METACAM NDV, Boeringher, Ingelheim am Rhein, Germany) was injected at the end of the procedure, and the animal was exubated. All surgeries were identically performed: the mandibuletomies and the microvascular reconstructions were carried out by the same surgeon while a second harvested and seeded the TBM into the BCP implants.

### 2.4.4. Postoperative management

Intramuscular Meloxicam was administered daily for 5 days (METACAM 0.5 mg kg⁻¹, Zoetis) and fentanyl patches for 3 days (DUROGESIC NDV, 100 µg h⁻¹, Janssen-Cilag, Beerse, Belgium) as analgesic treatment. Amoxicillin-clavulanic acid was also provided 8 days postoperatively (SYNULOX oral suspension, 1 mL 20 kg⁻¹, Zoetis). Feeding consisted mainly of hay and wet granules in the first days, then animals returned to a normal diet after two weeks.

Euthanasia of sheep was achieved by IV overdose of sodium pentorbital (20 mL of DOLETHAL NDV, Vétoquinol, Magny-Vernois, France). It was scheduled after 3 months of implantation for Sh1-5 and Sh7-11, and 12 months for Sh6 and Sh12. The permeability of the AVL was immediately assessed postmortem by irrigation of the TFA using a flexible catheter (22 gauges) and saline solution.

## 2.5. Computed tomography analysis

CT scans were performed after the removal of the rigid fixation plate (2.7 mm) from the hemimandible (Aquilion 16, Toshiba, Tokyo, Japan; scanning parameters: tube voltage 120 kV, 120 mA, 0.5 mm slice) to assess bone formation and osseointegration of the axially vascularized BCP implant. Images were exported in DICOM format and analyzed via Horos software to perform multipli-

nar (MPR) and 3-dimensional (3D) reconstruction for morphological analyses.

## 2.6. Microcomputed tomography analysis

Micro-CT analysis of the BCP-based implants was conducted after their explantation from the half-mandibles. Qualitative analysis of the total mineral and newly formed bone contents was performed using a SkyScan-1272 high-resolution 3D X-ray micro-computed tomography (micro-CT) system for small sample imaging (Brucker, Belgium). The scanner was equipped with a 20 to 100 kV (10 W) X-ray source and an 11 mega-pixel X-ray detector. Each sample was placed on a holder with the sagittal suture oriented parallel to the X-ray detector and scanned using a 0.11 mm copper filter, 26 µm isotropic voxels, a 0.9° rotation step, and frame averaging of 2. For 3D reconstruction (NRecon software, Bruker) without smoothing, ring artifact correction, beam hardening correction and the absorption coefficient were set to 5 and 10% and from 0 to 0.04, respectively. Representative 3D images were created using CTvox software (Bruker) for each implant to illustrate bone formation.

Quantitative analyses were performed with DragonFly (ORS, Canada) using a deep learning module for realistic and accurate quantification of bone tissue (Fig. S4 in the Supporting Information). Dragonfly provides solutions for advanced image processing, segmentation, and quantification with higher accuracy compared to human expert executing the same task [57-60]. In short, a total of 8-10 cross-sections (over more than 2000 per sample) were selected every 200 cross-sections to represent the whole sample. On each selected cross-section, 2 to 5 random analysis zones, including bone, ceramic, and background (plus noise and artifacts), were manually segmented to train the segmentation routine. Augmentation of the size of the dataset was also virtually increased using translations, rotations, deformation (shear, 2° maximum) and contrast modification of these segmented zones. An input of patch size 32 was used for the optimization algorithm (Adadelta) with an adaptive learning rate (initially equal to 1), running with an Adam-Dicloss loss function and considering a batch size of 512. Convergence of the training was obtained before 200 epochs. The trained segmentation algorithm was checked on 10 random cross-sections, corrected if required, and then applied to the whole sample. To investigate the phenomena driving the regeneration of the mandibular defect, 8 different volumes of interest were defined (Fig. S4A in the Supporting Information).

## 2.7. Histology

Newly formed bone and osteoblastic cells were stained with hematoxylin eosin safran (HES) and Movat pentachrome. In short, specimens were fixed for 5 days in 4% paraformaldehyde and then dehydrated through immersion in a graded series of ethanol. Nondecalcified bone specimens were infiltrated and embedded in glycol-methyl-methacrylate (GMMA; Technovit 9100, Kulzer, Germany). A sagittal section was obtained in the vascular gutter axis of each implant using a precision saw (Isomet 1000; Buehler, Uzwill, Swiss), and serial 5 µm sections were cut using a hard tissue microtome (Polycut SM 2500; Leica, Wetzlar, Germany). Sections were stained with HES and Movat pentachrome and then examined using a light microscope (Leica-DM 4000 B, Wetzlar, Germany).

## 2.8. Scanning electron microscopy

After embedding in resin and processing as described in Section 2.7, histological sections of the samples were sput-
For the experimental group (Sh1-Sh6), the BCP-based implant was first loaded with 5 mL of TBM (Fig. S3B in the Supporting Information) as described in Section 4.5.2. and placed into the SMD. A second plate (1.5 mm, Stryker France, Lyon, France) fixed at the basal edge of the scaffold using 2 ten-millimeter screws (Fig. S3C in the Supporting Information) was used to limit eventual motions of the implant under loading (e.g., mastication). Three minutes after an intravenous injection of heparin (50 UI kg⁻¹, HEPARINE CHOAY NDH, Sanofi-Aventis, Paris, France), the FTA and the FV were anastomosed under a microsurgical magnifier (8.0 polypropylene, Prolene, Ethicon) to create the AVL, which was diverted in the implant gutter (Fig. S3D in the Supporting Information).

For the clinical control group (Sh7-Sh12), reconstruction of the SMD was performed with an osseous vascularized free flap. In short, the iliac crest (ipsilateral side of the SMD) was exposed after incision of the skin and gentle dissection of the muscles (i.e., gluteus medius, tensor fascia latae and rectus abdominis). The DCIP was dissected up to the common iliac pedicle. Osteotomy of the iliac crest was then performed to obtain a vascularized bone flap matching the mandible defect (≈35 mm long x ≈55 mm high, Fig. S2C in the Supporting Information). Three minutes after an intravenous injection of heparin (50 UI kg⁻¹), the bone flap was harvested (section of the DCIP) and then placed in the SMD and fixed (osteosynthesis plate and 12 mm screws). The DCIP vein and artery were anastomosed to the FV and TFA (8.0 polypropylene), respectively.

A patency test for both the AVL and the free flap anastomoses was conducted before closure of the operative sites.

For both groups, the masseter and medial pterygoid muscles were sutured to the 2.7 mm-thick fixation plate using Vicryl 2/0 sutures. The wound was closed by bilayer suturing of the skin using Vicryl 3.0 (Etichon). The wound was cleaned with physiological serum and covered with a dry dressing. Meloxicam (0.5 mg kg⁻¹, METACAM NDV, Boeringher, Ingelheim am Rhein, Germany) was injected at the end of the procedure, and the animal was exubated. All surgeries were identically performed: the mandibuletomies and the microvascular reconstructions were carried out by the same surgeon while a second harvested and seeded the TBM into the BCP implants.

### 2.4.4. Postoperative management

Intramuscular Meloxicam was administered daily for 5 days (METACAM 0.5 mg kg⁻¹, Zoetis) and fentanyl patches for 3 days (DUROGESIC NDV, 100 µg h⁻¹, Janssen-Cilag, Beerse, Belgium) as analgesic treatment. Amoxicillin-clavulanic acid was also provided 8 days postoperatively (SYNULOX oral suspension, 1 mL 20 kg⁻¹, Zoetis). Feeding consisted mainly of hay and wet granules in the first days, then animals returned to a normal diet after two weeks.

Euthanasia of sheep was achieved by IV overdose of sodium pentorbital (20 mL of DOLETHAL NDV, Vétoquinol, Magny-Vernois, France). It was scheduled after 3 months of implantation for Sh1-5 and Sh7-11, and 12 months for Sh6 and Sh12. The permeability of the AVL was immediately assessed postmortem by irrigation of the TFA using a flexible catheter (22 gauges) and saline solution.

## 2.5. Computed tomography analysis

CT scans were performed after the removal of the rigid fixation plate (2.7 mm) from the hemimandible (Aquilion 16, Toshiba, Tokyo, Japan; scanning parameters: tube voltage 120 kV, 120 mA, 0.5 mm slice) to assess bone formation and osseointegration of the axially vascularized BCP implant. Images were exported in DICOM format and analyzed via Horos software to perform multipli-

nar (MPR) and 3-dimensional (3D) reconstruction for morphological analyses.

## 2.6. Microcomputed tomography analysis

Micro-CT analysis of the BCP-based implants was conducted after their explantation from the half-mandibles. Qualitative analysis of the total mineral and newly formed bone contents was performed using a SkyScan-1272 high-resolution 3D X-ray micro-computed tomography (micro-CT) system for small sample imaging (Brucker, Belgium). The scanner was equipped with a 20 to 100 kV (10 W) X-ray source and an 11 mega-pixel X-ray detector. Each sample was placed on a holder with the sagittal suture oriented parallel to the X-ray detector and scanned using a 0.11 mm copper filter, 26 µm isotropic voxels, a 0.9° rotation step, and frame averaging of 2. For 3D reconstruction (NRecon software, Bruker) without smoothing, ring artifact correction, beam hardening correction and the absorption coefficient were set to 5 and 10% and from 0 to 0.04, respectively. Representative 3D images were created using CTvox software (Bruker) for each implant to illustrate bone formation.

Quantitative analyses were performed with DragonFly (ORS, Canada) using a deep learning module for realistic and accurate quantification of bone tissue (Fig. S4 in the Supporting Information). Dragonfly provides solutions for advanced image processing, segmentation, and quantification with higher accuracy compared to human expert executing the same task [57-60]. In short, a total of 8-10 cross-sections (over more than 2000 per sample) were selected every 200 cross-sections to represent the whole sample. On each selected cross-section, 2 to 5 random analysis zones, including bone, ceramic, and background (plus noise and artifacts), were manually segmented to train the segmentation routine. Augmentation of the size of the dataset was also virtually increased using translations, rotations, deformation (shear, 2° maximum) and contrast modification of these segmented zones. An input of patch size 32 was used for the optimization algorithm (Adadelta) with an adaptive learning rate (initially equal to 1), running with an Oracle-Diceloss loss function and considering a batch size of 512. Convergence of the training was obtained before 200 epochs. The trained segmentation algorithm was checked on 10 random cross-sections, corrected if required, and then applied to the whole sample. To investigate the phenomena driving the regeneration of the mandibular defect, 8 different volumes of interest were defined (Fig. S4A in the Supporting Information).

## 2.7. Histology

Newly formed bone and osteoblastic cells were stained with hematoxylin eosin safran (HES) and Movat pentachrome. In short, specimens were fixed for 5 days in 4% paraformaldehyde and then dehydrated through immersion in a graded series of ethanol. Nondecalcified bone specimens were infiltrated and embedded in glycol-methyl-methacrylate (GMMA; Technovit 9100, Kulzer, Germany). A sagittal section was obtained in the vascular gutter axis of each implant using a precision saw (Isomet 1000; Buehler, Uzwill, Swiss), and serial 5 µm sections were cut using a hard tissue microtome (Polycut SM 2500; Leica, Wetzlar, Germany). Sections were stained with HES and Movat pentachrome and then examined using a light microscope (Leica-DM 4000 B, Wetzlar, Germany).

## 2.8. Scanning electron microscopy

After embedding in resin and processing as described in Section 2.7, histological sections of the samples were sput-
tered with gold (Sputter Coater Desk V Denton Vacuum) and then imaged using backscattered electrons at 20 kV with a Zeiss LEO 1450VP SEM. The whole surface of the explant was mapped, and the resulting mosaic of images was merged in a single high-resolution SEM image. Similar to the µCT analyses, deep learning algorithms were used for bone and bioceramic segmentation, significantly enhancing segmentation quality and reproducibility.

## 2.9. Statistical methods

Data are presented as representative images, representative experiments, or as means ± deviation, with N indicating the number of independent experiments. One-way analysis of variance (ANOVA) and Tukey post-hoc test were performed using StatPage calculator (http://statpages.info/anova1sm.html) whenever required and possible. For all analyses, differences at $p < 0.05$ were considered statistically significant.

# 3. Results

## 3.1. Customized implants

Macroscopic architectural features (i.e., gyroid porous structure, gutter, screw holes) of the produced biphasic calcium phosphate (BCP) implants matched the numerical model with a maximum deviation of ± 250 µm over the entire implant (Fig. 1A, B). Deviation from the theoretical CAD model followed a Gaussian distribution, centered on + 6 µm, with a full-width at half-maximum (FWHM) of 155 µm (Fig. 1C). These deviations are representative of the capability of the developed additive manufacturing process but also account for inaccuracies related to the imaging of the printed bioceramic and eventual errors during the numerical analysis, estimated to be approximately 50 µm. Mesoscopic cusps lower than 20 µm (depth and width, data not shown) within the (x,y) planes (with z the implant thickness), as a result of the manufacturing process [53], were hardly observed. The implant surface displayed a roughness at the microscale (1-10 µm) evaluated to be under 2 µm and approximately 10% s/s of submicronic pores (N=5) (Fig. 1B), as observed previously on BCP-based disks [52]. The composition of the BCP implants was assessed at 59.4 ± 0.3% (w/w) hydroxyapatite (HA) and 40.6 ± 0.3% (w/w) β-tricalcium phosphate (β-TCP) by means of a standard procedure based on X-ray diffraction patterns (N=3). More information about the XRD procedure is detailed in Paré et al. [52]. No other crystalline or amorphous phase was detected.

## 3.2. Clinical follow-up

All animals survived the surgical procedure with an average surgery time of 297 ± 8 min and 193 ± 12 min for the clinical control group (iliac crest VBT) and experimental group (BM loaded scaffold + AVL), respectively. Although surgical procedures were successful, a revision of the venous anastomosis to restore the blood flow was systematically required perioperatively in the control group (Sh7 to Sh9) that had the SMD treated with VBT. These sheep (Sh7 to Sh9) then displayed cheek swelling with purulent discharge and fever 10 days postoperatively. As no significant improvement was observed after 14 days despite antibiotic therapy, animals were euthanized in accordance with ethical considerations. In all cases, the explanted VBTs showed complete necrosis of the muscles covering the transplanted bone as well as a thrombosis of the venous anastomosis (Fig. S5 in the Supporting Information). In view of the complexity of the surgery for the animals

(e.g., donor site morbidity, duration of the procedure) and this systematic thrombosis, further experiments to repair the SMD with vascularized free flaps were aborted (Sh10 to Sh12) following animal welfare guidelines.

On the other hand, no major complications were observed with sheep in the experimental group, and so the experiment was carried out as planned (Sh1 to Sh5 and Sh6 euthanized after 3 and 12 months, respectively). All sheep returned to oral feeding the day of the surgical procedure and had a restoration of the mandible contour. During the healing period, no reconstruction or plate exposure occurred, and no abnormal findings such as inflammation, infection or separation in the surgical wound were observed for Sh1, Sh2, Sh3, Sh4 and Sh6. Sh5 had ipsilateral cheek inflammation for the first few days, without purulent discharge or fever. Antibiotic prophylaxis was prolonged for 7 days, allowing complete recovery. However, further postsacrifice analyses (Section 2.4) revealed an isolated abscess encapsulated into the masseter muscle. Whether it was for the 3-month or 12-month experimental groups, all the AVLs were permeable at the sacrifice time-point.

## 3.3. CT scan

The multiplanar and 3D reconstructions showed well-positioned scaffolds and anatomical reconstruction of the angle region in all cases (Fig. 2). Implant fractures were systematically observed; however, no displacement, loss of integrity or spread of materials into the surrounding soft tissues were reported. However, while the morphology of the Sh1, Sh2, Sh3, Sh5 and Sh6 implants was mostly preserved, the Sh4 implant was widely fragmented.

At the 3-month (Sh1 to Sh5) time point, CT images revealed evident bone ingrowth from the defect edges toward the center of the implants, partially covering the medial (i.e., pterygoid region) and lateral (i.e., masseter region) implant surfaces, particularly for Sh1, Sh2, and Sh4. Indeed, these three implants displayed close contact with the SMD edges and were embedded in a bony matrix that invaded the implant macroporosity to create a bridging of the defect on its entire length locally. For Sh3 and Sh5, poorer contact between the caudal edge of the SMD and the implant hindered proper osseointegration on this side; nonetheless, bone formation still occurred on the rostral side and within the implant macroporosity.

At 12 months (Sh6), the implant had wide osseointegration at both the rostral and caudal edges, allowing complete bridging of the SMD. A homogenous bone apposition was visible at the whole implant surface (i.e., external and internal networks). Cracks respecting the overall shape of the implant were also observed with no ectopic spread of the materials.

## 3.4. Micro-CT

Qualitative CT observations were corroborated and refined with quantitative µCT analyses. The benefits of using deep-learning routines for image segmentation (versus standard manual thresholding) was demonstrated, as illustrated in supplementary Fig. S6. Consequently, µCT and SEM data were quantitatively analyzed with DragonFly's deep-learning module (Figs. 3-5). As a direct consequence of implant fragmentation, the analysis volume $V_a$ (Fig. 3A), considered for bone quantification, was higher than the initial volume $V_i$ of the SMD (volume expansion of 21.6 ± 7.6%, Fig. 3B). As anticipated, biodegradation of BCP implants was relatively low, as 90.7 ± 1.1% and 75.5% of the implant remained after 3 and 12 months of implantation, respectively (Fig. 3C).
tered with gold (Sputter Coater Desk V Denton Vacuum) and then imaged using backscattered electrons at 20 kV with a Zeiss LEO 1450VP SEM. The whole surface of the explant was mapped, and the resulting mosaic of images was merged in a single high-resolution SEM image. Similar to the µCT analyses, deep learning algorithms were used for bone and bioceramic segmentation, significantly enhancing segmentation quality and reproducibility.

## 2.9. Statistical methods

Data are presented as representative images, representative experiments, or as means ± deviation, with N indicating the number of independent experiments. One-way analysis of variance (ANOVA) and Tukey post-hoc test were performed using StatPage calculator (http://statpages.info/anova1sm.html) whenever required and possible. For all analyses, differences at $p < 0.05$ were considered statistically significant.

# 3. Results

## 3.1. Customized implants

Macroscopic architectural features (i.e., gyroid porous structure, gutter, screw holes) of the produced biphasic calcium phosphate (BCP) implants matched the numerical model with a maximum deviation of ± 250 µm over the entire implant (Fig. 1A, B). Deviation from the theoretical CAD model followed a Gaussian distribution, centered on + 6 µm, with a full-width at half-maximum (FWHM) of 155 µm (Fig. 1C). These deviations are representative of the capability of the developed additive manufacturing process but also account for inaccuracies related to the imaging of the printed bioceramic and eventual errors during the numerical analysis, estimated to be approximately 50 µm. Mesoscopic cusps lower than 20 µm (depth and width, data not shown) within the (x,y) planes (with z the implant thickness), as a result of the manufacturing process [53], were hardly observed. The implant surface displayed a roughness at the microscale (1-10 µm) evaluated to be under 2 µm and approximately 10% s/s of submicronic pores (N=5) (Fig. 1B), as observed previously on BCP-based disks [52]. The composition of the BCP implants was assessed at 59.4 ± 0.3% (w/w) hydroxyapatite (HA) and 40.6 ± 0.3% (w/w) β-tricalcium phosphate (β-TCP) by means of a standard procedure based on X-ray diffraction patterns (N=3). More information about the XRD procedure is detailed in Paré et al. [52]. No other crystalline or amorphous phase was detected.

## 3.2. Clinical follow-up

All animals survived the surgical procedure with an average surgery time of 297 ± 8 min and 193 ± 12 min for the clinical control group (iliac crest VBT) and experimental group (BM loaded scaffold + AVL), respectively. Although surgical procedures were successful, a revision of the venous anastomosis to restore the blood flow was systematically required perioperatively in the control group (Sh7 to Sh9) that had the SMD treated with VBT. These sheep (Sh7 to Sh9) then displayed cheek swelling with purulent discharge and fever 10 days postoperatively. As no significant improvement was observed after 14 days despite antibiotic therapy, animals were euthanized in accordance with ethical considerations. In all cases, the explanted VBTs showed complete necrosis of the muscles covering the transplanted bone as well as a thrombosis of the venous anastomosis (Fig. S5 in the Supporting Information). In view of the complexity of the surgery for the animals

(e.g., donor site morbidity, duration of the procedure) and this systematic thrombosis, further experiments to repair the SMD with vascularized free flaps were aborted (Sh10 to Sh12) following animal welfare guidelines.

On the other hand, no major complications were observed with sheep in the experimental group, and so the experiment was carried out as planned (Sh1 to Sh5 and Sh6 euthanized after 3 and 12 months, respectively). All sheep returned to oral feeding the day of the surgical procedure and had a restoration of the mandible contour. During the healing period, no reconstruction or plate exposure occurred, and no abnormal findings such as inflammation, infection or separation in the surgical wound were observed for Sh1, Sh2, Sh3, Sh4 and Sh6. Sh5 had ipsilateral cheek inflammation for the first few days, without purulent discharge or fever. Antibiotic prophylaxis was prolonged for 7 days, allowing complete recovery. However, further postsacrifice analyses (Section 2.4) revealed an isolated abscess encapsulated into the masseter muscle. Whether it was for the 3-month or 12-month experimental groups, all the AVLs were permeable at the sacrifice time-point.

## 3.3. CT scan

The multiplanar and 3D reconstructions showed well-positioned scaffolds and anatomical reconstruction of the angle region in all cases (Fig. 2). Implant fractures were systematically observed; however, no displacement, loss of integrity or spread of materials into the surrounding soft tissues were reported. However, while the morphology of the Sh1, Sh2, Sh3, Sh5 and Sh6 implants was mostly preserved, the Sh4 implant was widely fragmented.

At the 3-month (Sh1 to Sh5) time point, CT images revealed evident bone ingrowth from the defect edges toward the center of the implants, partially covering the medial (i.e., pterygoid region) and lateral (i.e., masseter region) implant surfaces, particularly for Sh1, Sh2, and Sh4. Indeed, these three implants displayed close contact with the SMD edges and were embedded in a bony matrix that invaded the implant macroporosity to create a bridging of the defect on its entire length locally. For Sh3 and Sh5, poorer contact between the caudal edge of the SMD and the implant hindered proper osseointegration on this side; nonetheless, bone formation still occurred on the rostral side and within the implant macroporosity.

At 12 months (Sh6), the implant had wide osseointegration at both the rostral and caudal edges, allowing complete bridging of the SMD. A homogenous bone apposition was visible at the whole implant surface (i.e., external and internal networks). Cracks respecting the overall shape of the implant were also observed with no ectopic spread of the materials.

## 3.4. Micro-CT

Qualitative CT observations were corroborated and refined with quantitative µCT analyses. The benefits of using deep-learning routines for image segmentation (versus standard manual thresholding) was demonstrated, as illustrated in supplementary Fig. S6. Consequently, µCT and SEM data were quantitatively analyzed with DragonFly's deep-learning module (Figs. 3-5). As a direct consequence of implant fragmentation, the analysis volume $V_a$ (Fig. 3A), considered for bone quantification, was higher than the initial volume $V_i$ of the SMD (volume expansion of 21.6 ± 7.6%, Fig. 3B). As anticipated, biodegradation of BCP implants was relatively low, as 90.7 ± 1.1% and 75.5% of the implant remained after 3 and 12 months of implantation, respectively (Fig. 3C).
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Standardized CaP-based implant for segmental mandibular bone defects in a sheep model. (A) Pictures and dimensions of a representative sample of implanted scaffolds. (B) CloudCompare analysis with images showing the superposition of a produced implant obtained using X-ray micro-CT analysis with its CAD model. Green indicates accurate dimensional matching with very little positive or negative deviation; blue and red indicate a lack or an excess of material, respectively. The 3D models of the produced implant match with the CAD model, with a deviation of ± 250 µm centered on 6 µm. (C) SEM images at different levels of magnification (x1 200, x5 000, x30 000) used to highlight details of the micro- and sub-microstructures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Due to this volume expansion of the implants (fragmentation related, $V_a>V_i$) and despite their moderate degradation, the ceramic part occupies 35% to 40% of $V_a$ after 3 or 12 months of implantation (Fig. 4A and 4B), leaving 60% to 65% of free space for the development of new bone tissue.

After 3 months of implantation, this free space was filled at 19.9, 22.0, 5.6, 28.9 and 15.3% with mineralized new bone tissue for Sh1 – Sh5 (Fig. 4C), respectively, which corresponds to an av-

erage of 18.3 ± 8.7% (23.6 ± 4.8% when excluding the sheep Sh3 and Sh5 which were affected by a poor bone/implant contact and treated for infection, respectively). After 12 months, bone occupied more than one-third (35.7%) of the available porous volume, and the remaining volume was filled with nonmineralized tissue (Fig. 4C).

As illustrated in Fig. 5, no significant difference in bone distribution was observed between the caudal and rostral sides of the
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Standardized CaP-based implant for segmental mandibular bone defects in a sheep model. (A) Pictures and dimensions of a representative sample of implanted scaffolds. (B) CloudCompare analysis with images showing the superposition of a produced implant obtained using X-ray micro-CT analysis with its CAD model. Green indicates accurate dimensional matching with very little positive or negative deviation; blue and red indicate a lack or an excess of material, respectively. The 3D models of the produced implant match with the CAD model, with a deviation of ± 250 µm centered on 6 µm. (C) SEM images at different levels of magnification (x1 200, x5 000, x30 000) used to highlight details of the micro- and sub-microstructures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Due to this volume expansion of the implants (fragmentation related, $V_a>V_i$) and despite their moderate degradation, the ceramic part occupies 35% to 40% of $V_a$ after 3 or 12 months of implantation (Fig. 4A and 4B), leaving 60% to 65% of free space for the development of new bone tissue.

After 3 months of implantation, this free space was filled at 19.9, 22.0, 5.6, 28.9 and 15.3% with mineralized new bone tissue for Sh1 – Sh5 (Fig. 4C), respectively, which corresponds to an av-

erage of 18.3 ± 8.7% (23.6 ± 4.8% when excluding the sheep Sh3 and Sh5 which were affected by a poor bone/implant contact and treated for infection, respectively). After 12 months, bone occupied more than one-third (35.7%) of the available porous volume, and the remaining volume was filled with nonmineralized tissue (Fig. 4C).

As illustrated in Fig. 5, no significant difference in bone distribution was observed between the caudal and rostral sides of the
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Mandible CT scan assessing bone healing at 3 and 12 months. At the 3-month assessment using axial, sagittal and 3D reconstruction images of the 5 custom-made implants show osteointegration and new bone covering the lateral surface (i.e., masseter region) in Sh1, 2, and 4 with abundant bone formation within, particularly in Sh4 and non-osseointegration of the Sh3 and Sh5 implants, the latter displaying periosteal apposition and spiculated bone formation. An explant at 12 months (Sh6) shows osseointegration at both the rostral and caudal edges, implant integrity and bone formation within its macroporosity. Abbreviations: Sh, sheep; Bo, new mature bone; OI, osseointegration; NOI, non-osseointegration; PA, periosteal apposition; SB, spiculated bone formation.</figcaption></figure>

implants. However, bone formation occurred preferentially on the pterygoid side (i.e., mouth) and middle part of the implant versus the masseter side (i.e., cheek).

### 3.5. SEM and histological analyses

SEM analyses of the samples confirmed the low biodegradation of the BCP implants, even after 12 months of implantation, although implant fracture occurred to a greater or lesser extent (Fig. 6). For the early timepoint, implant fracture seemed to be correlated to the amount of new bone formed: the more regeneration of the SMD there was, the less implant remained (e.g., Sh3 versus Sh4, Fig. 6A, B) and conversely. Cracks within

the implant did not lead to its collapse or the migration of BCP particles outside the defect and were partially (at 3 months) or totally (at 12 months) filled by new mineralized tissue. At 3 months, trabecular-like mineralized bone tissue was present within the bioceramic pores, whereas cortical-like bone was observed mainly in contact with the walls (Fig. 6A). As shown in Fig. 6C, 11.1 ± 4.0% of the implant surface was covered by new bone, with a higher proportion in Sh2 and Sh4 (15.3% and 14.7%, respectively). After 12 months of implantation (Sh6), trabecular-like bone was not observed, and dense lamellar bone covered 100% of the bioceramic surfaces (Fig. 6C); bone occupied 27.0% of the free space versus 10.0 ± 8.0% at 3 months (Fig. 6B).
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Mandible CT scan assessing bone healing at 3 and 12 months. At the 3-month assessment using axial, sagittal and 3D reconstruction images of the 5 custom-made implants show osteointegration and new bone covering the lateral surface (i.e., masseter region) in Sh1, 2, and 4 with abundant bone formation within, particularly in Sh4 and non-osseointegration of the Sh3 and Sh5 implants, the latter displaying periosteal apposition and spiculated bone formation. An explant at 12 months (Sh6) shows osseointegration at both the rostral and caudal edges, implant integrity and bone formation within its macroporosity. Abbreviations: Sh, sheep; Bo, new mature bone; OI, osseointegration; NOI, non-osseointegration; PA, periosteal apposition; SB, spiculated bone formation.</figcaption></figure>

implants. However, bone formation occurred preferentially on the pterygoid side (i.e., mouth) and middle part of the implant versus the masseter side (i.e., cheek).

### 3.5. SEM and histological analyses

SEM analyses of the samples confirmed the low biodegradation of the BCP implants, even after 12 months of implantation, although implant fracture occurred to a greater or lesser extent (Fig. 6). For the early timepoint, implant fracture seemed to be correlated to the amount of new bone formed: the more regeneration of the SMD there was, the less implant remained (e.g., Sh3 versus Sh4, Fig. 6A, B) and conversely. Cracks within

the implant did not lead to its collapse or the migration of BCP particles outside the defect and were partially (at 3 months) or totally (at 12 months) filled by new mineralized tissue. At 3 months, trabecular-like mineralized bone tissue was present within the bioceramic pores, whereas cortical-like bone was observed mainly in contact with the walls (Fig. 6A). As shown in Fig. 6C, 11.1 ± 4.0% of the implant surface was covered by new bone, with a higher proportion in Sh2 and Sh4 (15.3% and 14.7%, respectively). After 12 months of implantation (Sh6), trabecular-like bone was not observed, and dense lamellar bone covered 100% of the bioceramic surfaces (Fig. 6C); bone occupied 27.0% of the free space versus 10.0 ± 8.0% at 3 months (Fig. 6B).
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Definition of the segmental mandibular defect volumes for the quantitative analysis. (A) Definition and illustration of the parameters used for the quantitative micro-CT analysis, (B) comparison of the volume used for micro-CT quantitative analysis and the theoretical volume of the bone defect – expansion due to the fragmentation of the implant – and (C) scaffold biodegradation after 3 months (Sh1 to Sh5) and 12 months of implantation (Sh6).</figcaption></figure>

Notable differences were observed by histology (HES, Movat pentachrome) between the 3- and 12-month samples (Fig. 7).

At the 3-month timepoint, mostly immature woven bone was observed within the implant, sometimes forming bridges between the pores. Cartilaginous tissue was observed in contact with the mineralized new bone tissue (Fig. 7A, B), and implant porosity was mainly filled by a disorganized extracellular matrix (i.e., non-mineralized tissue in Fig. 7A). Bone marrow niches were not detected at 3 months, regardless of the analyzed sample. Vascularization invaded the whole sample, with the presence of capillaries but also larger blood vessels, sometimes entrapped within the mineralized matrix (Fig. 7A, e.g., Sh2 and Sh4). For Sh5, infection markers (e.g., plasma cell infiltration and micro abscess) were observed, confirming delayed bone formation (Fig. 7A).

After 12 months of implantation, the integrity of the implant was preserved (i.e., shape and macroarchitecture) and complete

bridging of the SMD with wide osseointegration at the SMD edges. The entire implant surface was covered by a layer of lamellar matrix bone of varying thickness where osteoid tissue could also be observed (Fig. 7C). Cartilaginous tissue was no longer present, and the disorganized extracellular matrix was replaced by a dense network of unilocular adipocytes (Fig. 7C).

## 4. Discussion

In this study, the regeneration of a massive critical size defect (i.e., SMD) with a VBT (standard of care) or a tissue engineered construct, combining a customized BCP-based implant, autologous total bone marrow and an arteriovenous loop, was assessed in a clinically relevant model. Indeed, most of the investigations performed on "human-sized" animal models (e.g., sheep, goat, minipig) suffer from major flaws. These animal models are in-
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Definition of the segmental mandibular defect volumes for the quantitative analysis. (A) Definition and illustration of the parameters used for the quantitative micro-CT analysis, (B) comparison of the volume used for micro-CT quantitative analysis and the theoretical volume of the bone defect – expansion due to the fragmentation of the implant – and (C) scaffold biodegradation after 3 months (Sh1 to Sh5) and 12 months of implantation (Sh6).</figcaption></figure>

Notable differences were observed by histology (HES, Movat pentachrome) between the 3- and 12-month samples (Fig. 7).

At the 3-month timepoint, mostly immature woven bone was observed within the implant, sometimes forming bridges between the pores. Cartilaginous tissue was observed in contact with the mineralized new bone tissue (Fig. 7A, B), and implant porosity was mainly filled by a disorganized extracellular matrix (i.e., non-mineralized tissue in Fig. 7A). Bone marrow niches were not detected at 3 months, regardless of the analyzed sample. Vascularization invaded the whole sample, with the presence of capillaries but also larger blood vessels, sometimes entrapped within the mineralized matrix (Fig. 7A, e.g., Sh2 and Sh4). For Sh5, infection markers (e.g., plasma cell infiltration and micro abscess) were observed, confirming delayed bone formation (Fig. 7A).

After 12 months of implantation, the integrity of the implant was preserved (i.e., shape and macroarchitecture) and complete

bridging of the SMD with wide osseointegration at the SMD edges. The entire implant surface was covered by a layer of lamellar matrix bone of varying thickness where osteoid tissue could also be observed (Fig. 7C). Cartilaginous tissue was no longer present, and the disorganized extracellular matrix was replaced by a dense network of unilocular adipocytes (Fig. 7C).

## 4. Discussion

In this study, the regeneration of a massive critical size defect (i.e., SMD) with a VBT (standard of care) or a tissue engineered construct, combining a customized BCP-based implant, autologous total bone marrow and an arteriovenous loop, was assessed in a clinically relevant model. Indeed, most of the investigations performed on "human-sized" animal models (e.g., sheep, goat, minipig) suffer from major flaws. These animal models are in-
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Micro-CT analysis of bone healing and bone formation at 3 and 12 months. (A) 3D reconstruction of the custom-made implants assessing the osseointegration and the newly formed bone in the constructs. Lateral (i.e., masseter region) and axial views are represented at top and bottom respectively. New bone is represented in red, and the biomaterial in turquoise blue. Images show notable new bone both at the rostral and caudal edges and in the upper gutter area in Sh2 and Sh4, new bone at the caudal edge and in the upper gutter area in Sh1, and low bone formation in Sh3 and Sh5. (B) Graph showing the quantitative analysis of the mineral tissue (i.e., new bone and BCP) into the total bone defect volume, with systematically more BCP than new bone at 3 months and 12 months, higher bone formation in Sh1, Sh2, Sh4 (11.6,13.3 and 19.0, respectively) than in Sh3 and Sh5 at 3 months, and 22.5% of new bone within the defect at 12 months (Sh6). (C) Graph showing the proportion of new bone and nonmineralized tissue (NMT) in the implant macroporosity with an average bone formation of 18.3 ± 8.7% at 3 months and 35.7% at 12 months. Abbreviation: Sh, Sheep; $V_a$, analysis volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

dubitably relevant to study bone [49,61], but the defects to repair are nonsegmental, nonmechanically stressed, and often of small size [62–69], which simplifies the experimental setup and surgery, and decreases experimental costs (e.g., fixation system). The clinical relevance of such experimental models has to be questioned; hence, the development of a massive SMD model for this study is comparable to real-case scenarios [5].

Rather than oversimplifying the animal model and consequently the pertinence of the study, an optimization of the “clinical workflow” was performed to ease the procedure [70]. Thus, the surgical strategy was refined by a cadaveric study conducted before any surgical intervention. Custom scaffolds with a gyroid macroporous architecture, known to be beneficial for bone regeneration [52,70], were specifically designed with a gutter to host the AVL, screw and thread holes to simplify the surgeon's work, and smoothed angles to avoid injuring animals. A 3D printed polylactic acid template of the implant served after sterilization as cutting guides to generate the standardized SMD. This approach, which is acknowledged to be very beneficial (e.g., surgical time, comfort, blood loss, recovery) [71,72], allowed for a surgical procedure 1/3 faster for the experimental group than for the gold standard (193 versus 297 min), limiting potential complications [73]. Furthermore, the clinical group, treated using the human standard of care (i.e., VBT), was aborted and all the sheep transplanted with

such a vascularized iliac crest were euthanized at D14 for obvious ethical and animal welfare reasons. Indeed, despite the high reliability of VBT procedure in human clinics (i.e., more than 95% of success) [1,5], we observed unusual modifications of the soft tissues after sectioning of the deep circumflex iliac pedicle (DCIP) in this animal model which clearly precluded satisfying surgical conditions to perform the venous anastomosis. The ischemia (i.e., time between the artery sectioning and its anastomosis) was similar as in clinical routine but it systematically caused a fast dryness of the fat around the deep circumflex iliac vein (DCIV), making the DCIV hardly dissectable and suturable. In addition, the fast return to normal feeding (i.e., mastication and lateral movements of the mandible) the day of the surgical procedure induced early mechanical constraints on the DCIP, that were likely noxious for the patency of venous anastomoses. Consequently, these issues probably explain why venous thrombosis systematically occurred and caused the necrosis of the VBT (i.e., iliac bone with the muscle covering it) [74] then leading to an infection of the transplant and surrounding tissues. Conversely, the AVL anastomoses with a higher blood pressure did not display such a complication, and animals treated with this vascularized bone construct returned quickly to normal activity until the end of the follow-up period. Another way to interpret these results is that the chosen SMD model was large enough to not be repaired by conventional autologous bone grafts
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Micro-CT analysis of bone healing and bone formation at 3 and 12 months. (A) 3D reconstruction of the custom-made implants assessing the osseointegration and the newly formed bone in the constructs. Lateral (i.e., masseter region) and axial views are represented at top and bottom respectively. New bone is represented in red, and the biomaterial in turquoise blue. Images show notable new bone both at the rostral and caudal edges and in the upper gutter area in Sh2 and Sh4, new bone at the caudal edge and in the upper gutter area in Sh1, and low bone formation in Sh3 and Sh5. (B) Graph showing the quantitative analysis of the mineral tissue (i.e., new bone and BCP) into the total bone defect volume, with systematically more BCP than new bone at 3 months and 12 months, higher bone formation in Sh1, Sh2, Sh4 (11.6,13.3 and 19.0, respectively) than in Sh3 and Sh5 at 3 months, and 22.5% of new bone within the defect at 12 months (Sh6). (C) Graph showing the proportion of new bone and nonmineralized tissue (NMT) in the implant macroporosity with an average bone formation of 18.3 ± 8.7% at 3 months and 35.7% at 12 months. Abbreviation: Sh, Sheep; $V_a$, analysis volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

dubitably relevant to study bone [49,61], but the defects to repair are nonsegmental, nonmechanically stressed, and often of small size [62–69], which simplifies the experimental setup and surgery, and decreases experimental costs (e.g., fixation system). The clinical relevance of such experimental models has to be questioned; hence, the development of a massive SMD model for this study is comparable to real-case scenarios [5].

Rather than oversimplifying the animal model and consequently the pertinence of the study, an optimization of the “clinical workflow” was performed to ease the procedure [70]. Thus, the surgical strategy was refined by a cadaveric study conducted before any surgical intervention. Custom scaffolds with a gyroid macroporous architecture, known to be beneficial for bone regeneration [52,70], were specifically designed with a gutter to host the AVL, screw and thread holes to simplify the surgeon's work, and smoothed angles to avoid injuring animals. A 3D printed polylactic acid template of the implant served after sterilization as cutting guides to generate the standardized SMD. This approach, which is acknowledged to be very beneficial (e.g., surgical time, comfort, blood loss, recovery) [71,72], allowed for a surgical procedure 1/3 faster for the experimental group than for the gold standard (193 versus 297 min), limiting potential complications [73]. Furthermore, the clinical group, treated using the human standard of care (i.e., VBT), was aborted and all the sheep transplanted with

such a vascularized iliac crest were euthanized at D14 for obvious ethical and animal welfare reasons. Indeed, despite the high reliability of VBT procedure in human clinics (i.e., more than 95% of success) [1,5], we observed unusual modifications of the soft tissues after sectioning of the deep circumflex iliac pedicle (DCIP) in this animal model which clearly precluded satisfying surgical conditions to perform the venous anastomosis. The ischemia (i.e., time between the artery sectioning and its anastomosis) was similar as in clinical routine but it systematically caused a fast dryness of the fat around the deep circumflex iliac vein (DCIV), making the DCIV hardly dissectable and suturable. In addition, the fast return to normal feeding (i.e., mastication and lateral movements of the mandible) the day of the surgical procedure induced early mechanical constraints on the DCIP, that were likely noxious for the patency of venous anastomoses. Consequently, these issues probably explain why venous thrombosis systematically occurred and caused the necrosis of the VBT (i.e., iliac bone with the muscle covering it) [74] then leading to an infection of the transplant and surrounding tissues. Conversely, the AVL anastomoses with a higher blood pressure did not display such a complication, and animals treated with this vascularized bone construct returned quickly to normal activity until the end of the follow-up period. Another way to interpret these results is that the chosen SMD model was large enough to not be repaired by conventional autologous bone grafts
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Segmentation and statistical analysis of bone distribution in the segmental mandibular defect. (A) Graphs showing no difference in bone formation between rostral, middle and caudal areas of the defect. (B) Significantly higher bone formation is observed in the middle region than in the pterygoid or masseter region. Right graphs combine results obtained on the experimental group (Sh1-6).</figcaption></figure>

(i.e., nonvascularized) [5], and was a relevant preclinical model in sheep mimicking real life scenarii of segmental mandibular defect. Unfortunately, even though VBT is the gold standard procedure in clinics, vascularized iliac crest transplant appeared unsuitable in sheep model, and cannot consequently be used as a positive control. To quote de Lacerda Schickert et al. [75] and extrapolate their remarks for load-bearing bone defects: "further standardization of preclinical studies is essential for reliable evaluation" of clinically relevant strategy for segmental mandibular defect.

Multimodal analyses revealed that regeneration of the defect at 3 months was in its early stages, without any sign of bone necrosis, osteolysis or intervening soft tissue layer as observed by Nolff et al. [76]. Tissues were mainly composed of immature woven bone, disorganized extracellular matrix, blood vessels, or cartilaginous tissue. Further investigations are required to identify the ossification mechanism(s), and both endochondral and intermembranous ossification probably occur concurrently in diverse areas of the scaffold. However, the presence of cartilaginous tissue near mineralizing zones (absent in the 12-month implant) may indicate local endochondral ossification [77], possibly due to fracture of the implants and micromotions induced by sheep mastication [78,79]. Bone formation seemed highly affected by implant fracture and/or biodegradation, as seen for Sh1 and Sh4, where a low density of cracks within the bioceramic was concomitant with low bone

formation and vice versa. Discrepancies in the nature and extent of mechanical stimuli between fractured and unfractured implants (e.g., stress shielding, micromovements) as well as the space freed each time a fracture occurs may explain the different biological responses (e.g., Sh4 vs. the others). Furthermore, at 3 months, the new bone was homogeneously distributed throughout the porous volume of the implant (Figs. 5 and 7), and not only in contact with the ceramic (only 11.1 ± 4.0% of the implant surface was covered by new bone), indicating that bone growth within the implant was not only achieved by bone apposition (i.e., growth by forming new layers on the surface of pre-existing layers).

Between 3 and 12 months of implantation, complete modeling of the immature new bone structure occurred, resulting in the total embedding of the BCP scaffolds in a healthy vascularized, mature and lamellar bone matrix that is constantly evolving, as witnessed by the presence of osteoid tissue [80]. If not blood vessels and bone, most of the remaining space was homogeneously filled with unilocular adipocytes, which makes this structure very similar to the normal structure of mandibular bone. Indeed, in healthy mammals including humans, the medullar cavity of the mandibular bone is filled with hematopoietic (red) bone marrow at first, which is physiologically and totally converted to fatty bone marrow (also called "yellow bone marrow" or "bone marrow adipose tissue") as early as adolescence [81-83]. The presence of this non-
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Segmentation and statistical analysis of bone distribution in the segmental mandibular defect. (A) Graphs showing no difference in bone formation between rostral, middle and caudal areas of the defect. (B) Significantly higher bone formation is observed in the middle region than in the pterygoid or masseter region. Right graphs combine results obtained on the experimental group (Sh1-6).</figcaption></figure>

(i.e., nonvascularized) [5], and was a relevant preclinical model in sheep mimicking real life scenarii of segmental mandibular defect. Unfortunately, even though VBT is the gold standard procedure in clinics, vascularized iliac crest transplant appeared unsuitable in sheep model, and cannot consequently be used as a positive control. To quote de Lacerda Schickert et al. [75] and extrapolate their remarks for load-bearing bone defects: "further standardization of preclinical studies is essential for reliable evaluation" of clinically relevant strategy for segmental mandibular defect.

Multimodal analyses revealed that regeneration of the defect at 3 months was in its early stages, without any sign of bone necrosis, osteolysis or intervening soft tissue layer as observed by Nolff et al. [76]. Tissues were mainly composed of immature woven bone, disorganized extracellular matrix, blood vessels, or cartilaginous tissue. Further investigations are required to identify the ossification mechanism(s), and both endochondral and intermembranous ossification probably occur concurrently in diverse areas of the scaffold. However, the presence of cartilaginous tissue near mineralizing zones (absent in the 12-month implant) may indicate local endochondral ossification [77], possibly due to fracture of the implants and micromotions induced by sheep mastication [78,79]. Bone formation seemed highly affected by implant fracture and/or biodegradation, as seen for Sh1 and Sh4, where a low density of cracks within the bioceramic was concomitant with low bone

formation and vice versa. Discrepancies in the nature and extent of mechanical stimuli between fractured and unfractured implants (e.g., stress shielding, micromovements) as well as the space freed each time a fracture occurs may explain the different biological responses (e.g., Sh4 vs. the others). Furthermore, at 3 months, the new bone was homogeneously distributed throughout the porous volume of the implant (Figs. 5 and 7), and not only in contact with the ceramic (only 11.1 ± 4.0% of the implant surface was covered by new bone), indicating that bone growth within the implant was not only achieved by bone apposition (i.e., growth by forming new layers on the surface of pre-existing layers).

Between 3 and 12 months of implantation, complete modeling of the immature new bone structure occurred, resulting in the total embedding of the BCP scaffolds in a healthy vascularized, mature and lamellar bone matrix that is constantly evolving, as witnessed by the presence of osteoid tissue [80]. If not blood vessels and bone, most of the remaining space was homogeneously filled with unilocular adipocytes, which makes this structure very similar to the normal structure of mandibular bone. Indeed, in healthy mammals including humans, the medullar cavity of the mandibular bone is filled with hematopoietic (red) bone marrow at first, which is physiologically and totally converted to fatty bone marrow (also called "yellow bone marrow" or "bone marrow adipose tissue") as early as adolescence [81-83]. The presence of this non-
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** SEM analysis of the BCP implants. (A) SEM images of the six bone constructs with magnifications showing the mature bone (Bo, Gray), the BCP scaffolds (white) and the nonmineralized tissue (NMT, black). Notable scaffold alterations were observed in areas with Bo formation (see highest magnifications). Trabecular bone was observed to have implant porosity at 3 months (Sh1 to Sh5), while lamellar bone completely covered the inner scaffold surface at 12 months. (B) Graphs showing the proportion of residual BCP and newly formed tissue (i.e., Bo and NMT) in the segmental bone defect. (C) The proportion of contact between the new bone and the inner implant surface at 3 (11.1±4.0%) and 12 months (100%). Abbreviations: Sh, Sheep; Bo, mature bone; NMT, non-mineralized tissue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

hematopoietic yellow bone marrow can have also been favored by the injection prior implantation of TBM within the scaffold, which was extemporaneously harvested from the iliac crest of the sheep. Comparable results were reported by Fernandes et al. [84] who attempted to repaired tibial large bone defects in sheep using scaffolds seeded with bone mesenchymal stem cells. Finally, red to yellow marrow conversion can also be activated during times of metabolic stress [85], which is clearly the case in our study. Globally, this tendency observed at 12 months has to be confirmed in a more extensive pre-clinical study (N ≥ 5).

It may not be irrational to hypothesize that the gyroid architecture of the implant ("mimicking" bone trabecular structure) combined with fresh autologous total bone marrow (driving tissues regeneration) and an intrinsic vascularization (supporting the formation of new tissues through the creation of a vascular network), were responsible for such a successful biological response. Therefore, regeneration of massive SMDs with this surgical strategy and custom-made implants based on a triply periodic minimal surfaces macroporous structure seems very promising and easily applicable to the clinical setting. However, this study shows that the composition of the implant (60% of HA and 40% of β-TCP, w/w), combined with its low macropore density (40% v/v), allowed physiological but fragmented bone regeneration. Indeed, even if a continuum of new physiological tissues is observed in the whole volume of the massive SMD defect (e.g., osteoid tissue, yellow bone marrow, blood vessels), this one is fragmented due to the presence of the non-biodegraded bioceramic: about 40% of the bone volume defect is still occupied by the ceramic at 12 months. As a direct

consequence of this weak biodegradation, the quality and quantity of bone generated within the defect may not be sufficient to support the chewing load if the osteosynthesis plate were to be removed even 12 months after the reconstruction (e.g., risk of bone fracture).

This study confirmed also the relevance of the deep-learning-based algorithms trained to perform image analyses. These methods represent a significant improvement over traditional segmentation methods based on manual thresholding, which tend to be quite inaccurate depending on the size and nature of the analyzed sample [86,87].

The next step will involve a larger animal study, implementing lessons learned from this proof of concept. Rather than standardized custom implants and tools (e.g., cutting guides), a personalized approach will be developed for each animal to perfectly adjust the implant to the morphology of the bone defect borders and thus optimize SMD regeneration. Indeed, the slightest lack of contact between the implant and the defect edges will lead to delayed or poor osteointegration, as observed for the caudal side of the Sh3 and Sh5 implants. In addition, the implants will be manufactured with biomaterials exhibiting homogeneous and high-rate degradation relative to BCP, such as doped hydroxyapatites [88-91], CaP-based cements [92] or composites [93,94], coupled with a larger macroporous volume (50% to 60%), to free enough space and stimulate even more bone regeneration through degradation byproducts [70,95]. Thus, because of constraints on the composition and on the porosity, the mechanical weakness of the future implants should be overcome by adapting their design [70].
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** SEM analysis of the BCP implants. (A) SEM images of the six bone constructs with magnifications showing the mature bone (Bo, Gray), the BCP scaffolds (white) and the nonmineralized tissue (NMT, black). Notable scaffold alterations were observed in areas with Bo formation (see highest magnifications). Trabecular bone was observed to have implant porosity at 3 months (Sh1 to Sh5), while lamellar bone completely covered the inner scaffold surface at 12 months. (B) Graphs showing the proportion of residual BCP and newly formed tissue (i.e., Bo and NMT) in the segmental bone defect. (C) The proportion of contact between the new bone and the inner implant surface at 3 (11.1±4.0%) and 12 months (100%). Abbreviations: Sh, Sheep; Bo, mature bone; NMT, non-mineralized tissue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

hematopoietic yellow bone marrow can have also been favored by the injection prior implantation of TBM within the scaffold, which was extemporaneously harvested from the iliac crest of the sheep. Comparable results were reported by Fernandes et al. [84] who attempted to repaired tibial large bone defects in sheep using scaffolds seeded with bone mesenchymal stem cells. Finally, red to yellow marrow conversion can also be activated during times of metabolic stress [85], which is clearly the case in our study. Globally, this tendency observed at 12 months has to be confirmed in a more extensive pre-clinical study (N ≥ 5).

It may not be irrational to hypothesize that the gyroid architecture of the implant ("mimicking" bone trabecular structure) combined with fresh autologous total bone marrow (driving tissues regeneration) and an intrinsic vascularization (supporting the formation of new tissues through the creation of a vascular network), were responsible for such a successful biological response. Therefore, regeneration of massive SMDs with this surgical strategy and custom-made implants based on a triply periodic minimal surfaces macroporous structure seems very promising and easily applicable to the clinical setting. However, this study shows that the composition of the implant (60% of HA and 40% of β-TCP, w/w), combined with its low macropore density (40% v/v), allowed physiological but fragmented bone regeneration. Indeed, even if a continuum of new physiological tissues is observed in the whole volume of the massive SMD defect (e.g., osteoid tissue, yellow bone marrow, blood vessels), this one is fragmented due to the presence of the non-biodegraded bioceramic: about 40% of the bone volume defect is still occupied by the ceramic at 12 months. As a direct

consequence of this weak biodegradation, the quality and quantity of bone generated within the defect may not be sufficient to support the chewing load if the osteosynthesis plate were to be removed even 12 months after the reconstruction (e.g., risk of bone fracture).

This study confirmed also the relevance of the deep-learning-based algorithms trained to perform image analyses. These methods represent a significant improvement over traditional segmentation methods based on manual thresholding, which tend to be quite inaccurate depending on the size and nature of the analyzed sample [86,87].

The next step will involve a larger animal study, implementing lessons learned from this proof of concept. Rather than standardized custom implants and tools (e.g., cutting guides), a personalized approach will be developed for each animal to perfectly adjust the implant to the morphology of the bone defect borders and thus optimize SMD regeneration. Indeed, the slightest lack of contact between the implant and the defect edges will lead to delayed or poor osteointegration, as observed for the caudal side of the Sh3 and Sh5 implants. In addition, the implants will be manufactured with biomaterials exhibiting homogeneous and high-rate degradation relative to BCP, such as doped hydroxyapatites [88-91], CaP-based cements [92] or composites [93,94], coupled with a larger macroporous volume (50% to 60%), to free enough space and stimulate even more bone regeneration through degradation byproducts [70,95]. Thus, because of constraints on the composition and on the porosity, the mechanical weakness of the future implants should be overcome by adapting their design [70].
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** HES and MOVAT staining of the bone constructs at 3 and 12 months. (A) HES staining of the six bone constructs at a similar depth as the SEM analysis with two corresponding magnifications showing the BCP scaffold (gray, BCP), the newly formed tissues including mature bone (Bo, red-orange), cartilage (Cart, yellow) and fibrovascular tissue (Fvt, pink). At 3 months, the images show full osseointegration and an abundant amount of Bo both at the edges and in the gutter area (upper and middle parts) in Sh2 and Sh4, moderate osseointegration and bone formation in Sh1 and low osseointegration and bone formation in Sh3 and Sh5. The highest magnifications showed vessel formation with surrounding bone formation in the network of implant macroporosity (e.g., Sh2), contiguity between fibrovascular tissue, cartilage and bone formations (e.g., Sh1 and Sh3), mature bone formation (e.g., Sh4) and cell plasmocytic infiltration with microabscessation in Sh5. At 12 months (Sh6), a fully osseointegrated scaffold with lamellar bone covering the whole inner surface of the implant and a notable amount of adipocytes filling the rest of the porosity corresponding to yellow bone marrow (YBM) were observed. (B) Comparison between SEM, HES and MOVAT staining showing the presence of the endochondral ossification process (contiguity of cartilage and bone) as well as the tissues corresponding to the nonmineralized tissue previously analyzed by SEM and micro-CT (e.g., cartilage and fibrovascular tissue). (C) HES and MOVAT staining showing the lamellar bone covering the inner surface of the implant as well as the osteoid bone and the yellow bone marrow at 12 months. Abbreviations: Sh, Sheep; BCP, BCP scaffold; Cart, cartilage; Fvt, fibrovascular tissue; Bo, mature bone; V, vessel formation; NMT, nonmineralized tissue; P, plasmocytic infiltration; Ab, abscess; YBM, yellow bone marrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

**5. Conclusion**

From a clinical perspective, this study presents a compelling proof of concept showing that a synthetic biologics-free approach can lead to the regeneration of massive segmental mandibular defect (15 cm³) in a relevant large animal model which mimics real life scenarios encountered in human clinic. From a research point of view, this study is informative on many ways. First, the standard of care in human clinics, i.e. VBT, cannot serve as positive control for the repair of segmental mandibular defect in a sheep model. As such, the need to define a positive control for such a model remains. Second, the use of deep learning routines for image analyses should be systematized to prevent data misinterpretation. Finally, the total embedding of the BCP-based scaffolds in a

healthy vascularized, mature and lamellar bone matrix confirmed the biocompatibility of this type of scaffold for such an application, but also revealed the inadequacy of their degradation rate; 75% of the initial ceramic volume remaining after 12 months of implantation. As a consequence, a clinical alternative to CE-marked BCP biomaterials must be developed to reach the adequate bone formation/scaffold biodegradation balance. Moreover, interventions taking advantage of fully personalized scaffolds (rather than standardized as in this study) and surgical techniques should be encouraged to optimize bone regeneration. Further in-depth preclinical investigation, where the experimental designs and protocols will be refined based on the key points highlighted and described in this proof of concept, is essential to improve the chances of translational success.
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** HES and MOVAT staining of the bone constructs at 3 and 12 months. (A) HES staining of the six bone constructs at a similar depth as the SEM analysis with two corresponding magnifications showing the BCP scaffold (gray, BCP), the newly formed tissues including mature bone (Bo, red-orange), cartilage (Cart, yellow) and fibrovascular tissue (Fvt, pink). At 3 months, the images show full osseointegration and an abundant amount of Bo both at the edges and in the gutter area (upper and middle parts) in Sh2 and Sh4, moderate osseointegration and bone formation in Sh1 and low osseointegration and bone formation in Sh3 and Sh5. The highest magnifications showed vessel formation with surrounding bone formation in the network of implant macroporosity (e.g., Sh2), contiguity between fibrovascular tissue, cartilage and bone formations (e.g., Sh1 and Sh3), mature bone formation (e.g., Sh4) and cell plasmocytic infiltration with microabscessation in Sh5. At 12 months (Sh6), a fully osseointegrated scaffold with lamellar bone covering the whole inner surface of the implant and a notable amount of adipocytes filling the rest of the porosity corresponding to yellow bone marrow (YBM) were observed. (B) Comparison between SEM, HES and MOVAT staining showing the presence of the endochondral ossification process (contiguity of cartilage and bone) as well as the tissues corresponding to the nonmineralized tissue previously analyzed by SEM and micro-CT (e.g., cartilage and fibrovascular tissue). (C) HES and MOVAT staining showing the lamellar bone covering the inner surface of the implant as well as the osteoid bone and the yellow bone marrow at 12 months. Abbreviations: Sh, Sheep; BCP, BCP scaffold; Cart, cartilage; Fvt, fibrovascular tissue; Bo, mature bone; V, vessel formation; NMT, nonmineralized tissue; P, plasmocytic infiltration; Ab, abscess; YBM, yellow bone marrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

**5. Conclusion**

From a clinical perspective, this study presents a compelling proof of concept showing that a synthetic biologics-free approach can lead to the regeneration of massive segmental mandibular defect (15 cm³) in a relevant large animal model which mimics real life scenarios encountered in human clinic. From a research point of view, this study is informative on many ways. First, the standard of care in human clinics, i.e. VBT, cannot serve as positive control for the repair of segmental mandibular defect in a sheep model. As such, the need to define a positive control for such a model remains. Second, the use of deep learning routines for image analyses should be systematized to prevent data misinterpretation. Finally, the total embedding of the BCP-based scaffolds in a

healthy vascularized, mature and lamellar bone matrix confirmed the biocompatibility of this type of scaffold for such an application, but also revealed the inadequacy of their degradation rate; 75% of the initial ceramic volume remaining after 12 months of implantation. As a consequence, a clinical alternative to CE-marked BCP biomaterials must be developed to reach the adequate bone formation/scaffold biodegradation balance. Moreover, interventions taking advantage of fully personalized scaffolds (rather than standardized as in this study) and surgical techniques should be encouraged to optimize bone regeneration. Further in-depth preclinical investigation, where the experimental designs and protocols will be refined based on the key points highlighted and described in this proof of concept, is essential to improve the chances of translational success.
# Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Acknowledgements

This work was supported by the "Fondation de L'Avenir" [grant agreement no. AP-RM-17-017] and the "Fondation des Gueules Cassées" [grant agreement no. 66-2017].

# Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2022.09.071.

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Declaration of Competing Interest

The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements

This work was supported by the "Fondation de L'Avenir" [grant agreement no. AP-RM-17-017] and the "Fondation des Gueules Cassées" [grant agreement no. 66-2017].

Supplementary materials

Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2022.09.071.

References

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[2] P. Tessier, H. Kawamoto, J. Posnick, Y. Raulo, J.F. Tulasne, S.A. Wolfe, Compli-cations of harvesting autogenous bone grafts: a group experience of 20,000 cases, Plast. Reconstr. Surg. 116 (2005) 72S-73S discussion 92S-94S.

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