Ishihara et al. Journal of Orthopaedic Surgery and Research 2014, 9:98
http://www.josr-online.com/content/9/1/98

RESEARCH ARTICLE

Open Access

Simultaneous regeneration of full-thickness
cartilage and subchondral bone defects in vivousing a three-dimensional scaffold-free
autologous construct derived from high-density
bone marrow-derived mesenchymal stem cells
Kohei Ishihara1, Koichi Nakayama1,2*, Shizuka Akieda1, Shuichi Matsuda1 and Yukihide Iwamoto1

Abstract
Background: In recent years, several methods have been developed for repairing full-thickness cartilage defects
by tissue engineering using mesenchymal stem cells. Most of these use scaffolds to achieve sufficient thickness.
However, considering the potential influence of scaffolds on the surrounding microenvironment, as well as
immunological issues, it is desirable to develop a scaffold-free technique. In this study, we developed a novel
technique, a scaffold-free autologous construct derived from bone marrow-derived mesenchymal stem cells(BM-MSCs), and successfully use this technique to regenerate cartilage and subchondral bone to repair anosteochondral defect in rabbit knees.
Methods: BM-MSCs were isolated from bone marrow liquid aspirated from the iliac crest of rabbits. After expansion
in culture dishes and re-suspension in 96-well plates, the cells spontaneously aggregated into a spheroid-likestructure. The spheroids were loaded into a tube-shaped Teflon mold with a 5-mm height and maintained under
air-liquid interface conditions. These loaded spheroids fused with each other, resulting in a cylinder-shaped
construct made of fused cells that conformed to the inner shape of the mold. The construct was implanted into
an osteochondral defect in rabbit knees and histologically analyzed 24 and 52 weeks after implantation using
Wakitani’s scoring system.
Results: Both bone and cartilage were regenerated, maintaining a constant thickness of cartilage. The mean
histological score was 10 ± 1.7 in the 24-week group and 9.7 ± 0.6 in the 52-week group. There was no significant
difference between the 24- and 52-week groups in either parameter of the score, indicating that no deteriorationof the repaired tissue occurred during the intervening period.
Conclusions: Using our novel technique, which employs a three-dimensional scaffold-free autologous constructderived from BM-MSCs, we successfully achieved simultaneous regeneration of bone and cartilage for up to 1 year
in vivo. This method has potential for clinical use as a safe and effective method for repairing bone and cartilage defects.
Keywords: Tissue engineering, Mesenchymal stem cells, Osteochondral cartilage defect, Scaffold-free, Cartilage repair* Correspondence: nakayama@me.saga-u.ac.jp
1
Department of Orthopaedic Surgery, Graduate School of Medical Sciences,
Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka City, Fukuoka
812-8582, Japan2
Department of Advanced Technology Fusion, Graduate School of Scienceand Engineering, Saga University, 1 Honjo-machi, Saga City, Saga 840-8502,
Japan© 2014 Ishihara et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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Background
Repairing damaged cartilage, as in traumatic lesions of
the joint surface or degenerative damage due to osteoarthritis, remains a clinical challenge because of the poor
self-healing ability of cartilage [1]. Several existing treatments for the repair of damaged cartilage, including
mosaicplasty [2], microfracture [3], and autologous chondrocyte implantation (ACI) [4], have been used successfullyin patients to relieve pain and improve joint function.
However, the long-term results of mosaicplasty and microfracture are unsatisfactory because the replaced tissue is
not physiological hyaline cartilage, but mostly fibrous
cartilage, which lacks suitable mechanical properties[5,6]. Furthermore, in histological evaluations of therepaired tissue, ACI exhibited no significant differencecompared to microfracture in a randomized trial [7], andthe repaired tissue was predominantly fibrous cartilage.
Tissue engineering of functional articular cartilage
could help address these issues. In recent years, several
methods have been developed to repair full-thickness
cartilage defects. Some of these methods use synthetic
or biological scaffolds to achieve sufficient thickness,mechanical function, support cell attachment, migration,
proliferation, and differentiation [8,9]. However, these materials can influence the surrounding microenvironments[10] or cause immunological problems [11,12].
Several scaffold-free systems have also been investigated [13-16]. Without using a scaffold, however, it isdifficult to create enough thickness to fill in the defect.
A thickness of 5 mm is necessary to fill a full-thickness
articular cartilage defect in the knee. Furthermore, withor without using a scaffold, it is even more challenging forthe current methods to regenerate both articular cartilage
and subchondral bone in the context of an osteochondral
defect, which sometimes occurs in trauma, osteochondritis dissecans, osteonecrosis, or osteoarthritis.
Adherent cells grown in suspension form spheroids(aggregates) in order to avoid cell death. We confirmed
this phenomenon using bone marrow-derived mesenchymal stem cells (BM-MSCs). Furthermore, we developed anovel method for fusing spheroids derived from BMMSCs to make a large construct without using a scaffold.
Loading the spheroids into a molding chamber can produce a columnar structure consisting of fused spheroids;
we named this structure “high-density mesenchymal stem
cell, scaffold-free autologous construct” (HDMAC). We
hypothesized that HDMACs could be used to regenerate
both articular cartilage and subchondral bone in the context of an osteochondral defect. Furthermore, because
many other reports have achieved successful short-term
cartilage repair, we focused on achieving a relatively longterm result (up to 1 year) in an animal model.
The purposes of this study were to demonstrate ourmethod for making spheroids and a scaffold-free construct(HDMAC) using only undifferentiated BM-MSCs and toevaluate the 1-year outcome of simultaneous regenerationof cartilage and subchondral bone in an osteochondral
defect in rabbit knees.

Materials and methods
Rabbits

All experiments followed protocols approved by the institutional animal care and use committee (approval number:
A21-204-2). Fifteen mature female Japanese white rabbits(Japan Kyudo Inc, Tosu, Japan) with a mean weight of
3.9 kg (range 3.5–4.2) were eventually used for this study
after several pilot studies to establish the procedures indetail. Animals were housed in separate cages at the animal center of our institution with free access to water andstandard food. Two or three rabbits were euthanized at 3,
6, and 12 weeks and three rabbits were euthanized at eachof two time points (24 and 52 weeks) after implantation
for histological analysis.
Culture medium

Dulbecco’s modified Eagle’s medium (DMEM) (Nissui#05915, Nissui Pharmaceutical, Tokyo, Japan) containing
10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA),
100 U/mL penicillin, and 100 μg/mL streptomycin (SigmaAldrich, St. Louis, MO, USA) was used. No differentiation agents were used from cell isolation to constructformation.
Isolation and culture of mesenchymal stem cells (MSCs)

MSCs were isolated as described previously with somemodifications in the method [9]. Briefly, bone marrowliquid was aspirated from the iliac crest of the rabbits
under general anesthesia, and the obtained bone marrow
was mixed with heparin. After centrifugation at 1,000 rpm
for 5 min, the buffy coat of the bone marrow liquid was
aspirated and expanded into a 15-cm culture dish containing DMEM and 10% FBS. The cells obtained wereexpanded until they reached the necessary numbers(~4 × 107 cells).
Spheroid formation

Cells were detached by recombinant trypsin replacement,
re-suspended in DMEM plus 10% FBS, and then divided
into 96-well plates designed to prevent cells from adheringto the culture surface (Sumilon PrimeSurface, Sumitomo
Bakelite, Tokyo, Japan) at 4 × 104 cells per well. After incubation at 37°C and 5% CO2 for 2 days, the cells spontaneously aggregated into a spheroid-like structure (Figure 1a).
Construct formation

The spheroids described above were loaded into ahandmade tube-shaped Teflon mold with a diameter of
4.6 mm and a height of 5 mm (Figure 1b). At the bottom

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Figure 1 Manufacturing HDMACs. (a) BM-MSCs were isolated from bone marrow liquid aspirated from rabbits. After expansion, re-suspension,and incubation, the cells spontaneously aggregated into a spheroid-like structure. Each scale unit represents 1 μm. (b) A handmade tube-shaped
Teflon mold with a diameter of 4.6 mm and a height of 5 mm. The spheroids described in (a) were loaded into this mold. Each scale unit
represents 1 mm. (c) The tube-shaped mold was maintained under air-liquid interface conditions and incubated for a week. These loaded spheroids
fused with each other, and a cylinder-shaped construct made with fused cells was obtained according to the inner shape of the mold. Each scale unit
represents 1 mm. (d) Safranin O staining of HDMACs after 1 week of incubation. Scale bar =100 μm. (N = 1).

of the mold, a base disc was placed into the chamber; thebase disc was used for handling of the construct. Six
hundred to seven hundred spheroids were needed to makeone construct. The tube-shaped mold was maintained
under air-liquid interface conditions and incubated at
37°C and 5% CO2 for 1 week in DMEM plus 10% FBS.
These loaded spheroids fused with each other, resulting
in formation of a cylinder-shaped construct made of fused
cells that conformed to the inner shape of the mold(Figure 1c). We named these structures “high-density
mesenchymal stem cell, scaffold-free autologous constructs” (HDMACs).
Scanning electron microscopy (SEM)

Constructs were fixed with 1% glutaraldehyde/1.44% paraformaldehyde in buffer at 37°C for 60 min, frozen in liquid
nitrogen, and lyophilized. Next, samples were sputtercoated with an alloy of platinum and palladium and thenobserved by scanning electron microscopy (JSM 840A,
JEOL USA, Peabody, MA, USA).
Implantation in vivo

After general anesthesia, a cylindrical defect was created in the articular cartilage of the patella groove. The

diameter of the defect was set to 4.8 mm. The depth was
determined to be the same as the height of the cell
construct, usually 4–5 mm in depth, which reachedthe middle layer of the subchondral bone. The construct was gently implanted into the defect, the Teflon disc was removed, and the top of the construct
was flattened using the surgeon’s finger. No patches
were placed on the implant surface. The osteochondral defect in the contralateral knee was left empty
for controls. After washing and closure of the wound,the knee was immobilized with a cast for 1 week. Ina pilot study, individual rabbits were euthanized at 3,
6, and 12 weeks to confirm the early regeneration
process by histological evaluation. After obtaining positive
results from this pilot study, three knees were obtained ateach of two time points (24 and 52 weeks) and subjected
to histological analysis.
Micro-CT scanning

For morphological observation, the specimens were
scanned using a micro-CT (Hitachi-Aloka Medical, Tokyo,
Japan) after euthanasia. Sliced CT data were threedimensionally reconstructed (Real INTAGE, Kubota
Graphics Technology, Tokyo, Japan).

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Histological analysis and immunohistochemistry

Cell tracking

Specimens were fixed in 4% paraformaldehyde, decalcified with 0.5 M ethylenediaminetetraacetic acid (EDTA),
and embedded in paraffin wax. Sagittal sections (5 μm
thick) were stained with safranin O and Weigert’s iron
hematoxylin and then examined by light microscopy.
For immunohistochemistry, sections were pretreatedwith 0.4 mg/mL proteinase K (DAKO, Carpinteria, CA,
USA) for 10 min at room temperature. Endogenous peroxidases were quenched using 3% hydrogen peroxide inmethanol for 20 min at room temperature. The sections
were then incubated with mouse polyclonal antibody
against type II collagen (F-57 anti-hCL (II), FUJI Chemical
Industries, Toyama, Japan) and rabbit polyclonal antibody
against type X collagen (ab58632, Abcam, Cambridge,
MA, USA) at room temperature for 1 h. The immunostaining was performed using the VECTASTAIN ABC
reagent (Vector Laboratories, Burlingame, CA, USA) anddeveloped in 3,3’-diaminobenzidine and 0.02% H2O2 withhematoxylin counterstaining.

At first passage, cells were re-suspended at in DMEM
1.0 × 106 cells/mL and then were labeled with Qtracker
655 Cell Labeling Kit (Quantum Dot Corp., Hayward,
CA, USA).
The day after the cell labeling, the cells proceeded tospheroid formation as described above. HDMACs generated using the labeled cells were implanted in rabbit
knees as described above, and the rabbits were 6 weeks
later for histological examination.

Histological scoring

Histological findings were scored according to a scale
described by Wakitani et al. [9]. Sections were gradedaccording to 1) cell morphology (maximum 4 points), 2)
matrix staining (maximum 3 points), 3) surface regularity(maximum 3 points), 4) thickness of cartilage (maximum
2 points), and 5) integration of donor material into adjacent host cartilage (maximum 2 points). Scoring was
performed blindly by two orthopedic surgeons who wereindependent of this study, and the average of each score
was adopted.

Statistical analysis

For in vivo investigations, nonparametric comparisonswere performed using Student’s t test. P values lessthan 0.05 were considered to represent statisticalsignificance.

Results
Histology and scanning electron micrographs of HDMACs

Histological examination of HDMACs with safranin O
staining after 1 week of incubation revealed that thenucleus of cells were stained without fragmentation or
chromatin condensation at the whole area of the section,suggesting that the cells in the construct were viableand did not undergo apoptosis (Figure 1d). The matrix
was not stained red, suggesting that the cells in the
HDMACs had not been differentiated into chondrocytes
before the implantation. The ultrastructural morphologyof the HDMACs was assessed by SEM. The micrographs
revealed that these constructs contained flattened cellswith some amount of collagen fibers (Figure 2).

Figure 2 Scanning electron micrographs of HDMACs. SEMs showed that these constructs contained flattened cells (arrows and frames) withsome amount of collagen fibers. Original magnifications were (a) 430×, (b) 1,000×, and (c) 2,500×. Scale bar =10 μm. (N = 1).

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Pilot study for early repairing process in osteochondral
defectTo test our hypothesis that HDMACs would be able tosimultaneously regenerate cartilage and bone, two or
three rabbits were euthanized at 3, 6, and 12 weeks andmorphologically and histologically assessed (Figures 3a–i
and 4a–i). After 6 weeks of implantation, the surface ofthe defect was covered with cartilaginous white tissue.
Micro-CT scanning also suggested that bone formations
were present at the peripheral areas and developed toward
the central area. Histological examination suggested thatcartilaginous differentiation, which was detected by safranin O staining and immunostaining for type II collagens,occurred at peripheral areas of the implanted HDMACs
and developed toward the central area. At 6 weeks, all theimplanted area showed positive staining for safranin O
and type II collagen. At 12 weeks, endochondral boneformation progressed, and almost the whole area corresponding to the subchondral bone was replaced by bone

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structures. At the joint surface area of the implanted
tissue, hyaline cartilage was regenerated with a constant thickness.
By contrast, in a control rabbit without any implantation, the defect was filled with fibrous tissue. Althougha spontaneous repairing process was observed at the peripheral area that showed slight staining for safranin O ortype II collagen, the cartilage formation and the subchondral bone formation were incomplete at the central areaeven at 12 weeks (Figures 3j–l and 4j–l).
Cell tracking

HDMACs were made with labeled MSCs and implanted
into the osteochondral defect. Labeled cells were detected
as chondrocytes in the cartilage and osteocytes in the subchondral bone 6 weeks after implantation, suggesting thatthe implanted MSCs had spontaneously differentiated into
chondrocytes or osteocytes in accordance with their implanted positions (Figure 5).

Figure 3 Early repair processes in osteochondral defects. Gross appearance at (a) 3 weeks, (d) 6 weeks, and (g) 12 weeks. After 6 weeks of
implantation, the surface of the defect was covered with cartilaginous white tissue. Micro-CT scanning at (b, c) 3 weeks, (e, f) 6 weeks, and (h, i)
12 weeks suggested that bone formations were present at the peripheral areas and developed toward the central area. (j) Gross appearance and(k, l) micro-CT scanning of a control rabbit without any implantation at 12 weeks. Although the gross appearance showed that the defect wasfilled with white tissues, the subchondral bone formation was incomplete at the center of the defect at 12 weeks. Downwards arrows indicate
the borders of the defect. Representative subjects of two or three experiments for each time course are shown.

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Figure 4 Histological examination at early repair processes in osteochondral defects. Safranin O staining, immunostaining for type II
collagens, and IgG staining as controls at (a–c) 3 weeks, (d–f) 6 weeks, and (g–i) 12 weeks after the implantation. At 3 weeks, the implanted
tissue had not differentiated into hyaline cartilage yet. At 6 and 12 weeks, the joint surface area of the implanted tissue was stained with safranin
O and immunostained for type II collagen. Endochondral bone formation was observed at the peripheral area at 6 weeks and developed toward
the central area at 12 weeks. (j–l) A control rabbit without any implantation at 12 weeks. The defect was filled with fibrous tissue that did show aconstant staining for safranin O and immunostaining for type II collagen, especially at the surface and central areas. Scale bar = 1 mm. Downwards
arrows indicate the borders of the defect. Representative subjects of two or three experiments for each time course are shown.

Histological examination at 24 and 52 weeks

After obtaining the preliminary results regarding theearly regeneration process, we focused on morphologicaland histological evaluation at 24 and 52 weeks (n = 3each) (Figures 6a–j and 7a–h). CT scans revealed complete

regeneration of the subchondral bone. Histological examination revealed that hyaline cartilage, which was stained
for safranin O and immunostained for type II collagen, wasmaintained at a consistent thickness identical to that of thesurrounding original cartilage at both 24 and 52 weeks.

Figure 5 Cell tracking. HDMACs made with labeled MSCs were implanted to the osteochondral defect. The labeled cells were detected aschondrocytes in the cartilage and osteocytes in the subchondral bone 6 weeks after implantation, suggesting that the implanted MSCs had
spontaneously differentiated into chondrocytes or osteocytes in accordance with their implanted positions. Scale bar = 200 μm. (N = 1).

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Figure 6 Morphological and histological examination at 24 and 52 weeks. Gross appearance, micro-CT scanning, and safranin O staining at(a–e) 24 weeks and (f–j) 52 weeks. Micro-CT scans revealed complete regeneration of the subchondral bone. Histological examination revealed
that the regenerated cartilage was maintained with a consistent thickness identical to that of the surrounding original cartilage at both (d)24 weeks and (i) 52 weeks. Scale bar = 1 mm. (e, j) Tidemarks, which separate hyaline cartilage from the calcified cartilage, were seen (arrows).
Scale bar = 100 μm. (k–o) A control rabbit without any implantation at 52 weeks. The defect was mainly filled with fibrous tissue that did notexhibit formation of subchondral bone (m) or sufficient staining with safranin O (n). Although some area was stained with safranin O, tidemark
was not formed (o). Downwards arrows indicate the borders of the defect. Representative subjects of three experiments for each time course
are shown.

Immunostaining for type X collagens did not show positive staining in the implanted area during the regeneration
period. Tidemark, which separates hyaline cartilage fromthe calcified cartilage layer, was seen in these two groups,
suggesting the integrality of the regenerated articular
structures. By contrast, a control rabbit without any
implantation showed that the defect was mainly filled withfibrous tissue that did not exhibit the consistent staining
for safranin O or formation of the subchondral bone.

Although some area was stained with safranin O, tidemark
was not formed (Figure 6k–o).
The mean histological score was 10 ± 1.7 (standard
error (S.E.)) in the 24-week group and 9.7 ± 0.6 (S.E.)
in the 52-week group. There was no significant difference between the 24- and 52-week groups in either
parameter of the score, indicating that no deteriorationof the repaired tissue took place during this period(Table 1).

Figure 7 Immunohistochemistry at 24 and 52 weeks. Safranin O staining (a, e), immunostaining for type II collagens (b, f), immunostaining
for type X collagens (c, g), and IgG staining as controls (d, h) at 24 weeks (a–d) and 52 weeks (e–h). No positive Immunostaining for type X
collagens was observed in the implanted area during the regeneration period. Scale bar =200 μm. Representative subjects of three experiments
for each time course are shown.

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Table 1 Histological scores at 24 and 52 weeks after
implantation
24 weeks(n =3)

52 weeks(n =3)

p value

Cell morphology

2.7 (2–3)

2.7 (2–3)

N.S.

Matrix staining (metachromasia)

1.7 (1–2)

1.7 (1–2)

N.S.

Surface regularity

3

3

N.S.

Thickness of cartilage

1.3 (1–2)

1.7 (1–2)

N.S.

Integration of donor to
adjacent host cartilage

1.3 (1–2)

0.7 (0–1)

N.S.

Total score (score range 0–14)

10 (8–11)

9.7 (9–10)

N.S.

N.S. not significant.

Discussion
In this study, we demonstrated successful regenerationof articular cartilage and subchondral bone in an
osteochondral defect of rabbit knee joints using threedimensional HDMACs. The HDMACs were madewithout using any differentiation agents, such as growth
factors, and were implanted to the osteochondral defectwhile the cells in the construct were undifferentiated. The
implanted MSCs spontaneously differentiated into boneor cartilage, depending on the environment in which they
were implanted, and maintained the original border
between bone and cartilage. Eventually, by the 52-weekfollow-up, the simultaneous regeneration of bone andcartilage had been completed. The unique features of our
method are as follows: 1) A three-dimensional MSC
construct with sufficient thickness for bone and cartilage
repair can be made without a scaffold. 2) The construct is
implanted without induction of differentiation. 3) Simultaneous regeneration of bone and cartilage occurs
spontaneously by reproducing the differentiation process
involved in bone and cartilage formation. A major
strength of our study is that we could confirm that theregenerated bone and cartilage were maintained for up to
52 weeks.
However, one limitation of our study is that we did
not make an effort to increase the sample sizes for the
short-term results (e.g., within 12 weeks). After confirming in a few samples that MSCs spontaneously differentiate into bone or cartilage in vivo, as we originallyhypothesized, we placed greater emphasis on investigating the long-term results. We chose this course becausea large number of previous studies using other methods
had succeeded over the short term, but it remained that
deterioration of the repaired cartilage would occur overlong-term observation. Some previous studies using ascaffold reported frequent invasion of a bony structure
into the repaired cartilage, causing the cartilage to become thinner than the surrounding normal articularcartilage [9,17]. Furthermore, a scaffold has the potential
to influence surrounding microenvironments, which canPage 8 of 10

affect the regional specification of implanted MSCs
[10]. The presence of nonphysiological scaffolds could
interfere with the physiological distribution of signal
molecules, such as growth factors, that influence thedifferentiation of cells toward bone and cartilage.
Following a report of the efficacy of ACI by Brittberg
et al. [4], chondrocytes have been widely used in thefield of articular cartilage regeneration. The form of thegrafts used for implantation varies from monolayer culture [4] to tissue-engineered three-dimensional culturesin agarose [18] or atelocollagen gel [19]. A number ofclinical trials have been conducted, and some of them
reported successful outcomes in regard to patients’ symptoms or imaging endpoints [19,20]. However, it should be
noted that ACI was more effective in patients younger
than 45 years than in older patients [21]. In fact, the mean
age of clinical trials with satisfactory outcomes for relatively long follow-up periods ranges from 26.4 to 33.1 years
old [19,22]. This result may be related to the shortening oftelomeres in many adult cells, including chondrocytes[23]. Therefore, it may be necessary to use alternative
methods in middle-aged and elderly patients.
Bone and cartilage have the same origin, i.e., MSCs. In
a previous work, patient age made no difference in outcomes of methods using MSCs [21]. Therefore, MSCs
represent an attractive alternative to adult chondrocytes
as a cell source for bone and cartilage regeneration.
However, it is challenging to artificially control differentiation into the osteocyte or chondrocyte lineage after
implantation. The underlying molecular mechanism of
our method, in which a bone and articular cartilage
defect was repaired while maintaining the border between cartilage and subchondral bone, is currently unclear. However, cell tracking analysis revealed that bothcell types in the repaired tissues were derived dominantly
from the implanted MSCs within the construct rather
than the migrated cells from the surrounding tissues(Figure 5). Our HDMACs may mimic the cellular integration of embryonic osteogenesis and chondrogenesisthat originates from mesenchymal condensation. The
HDMACs were implanted without pretreatment to induce
differentiation. The micrographs by SEM revealed that the
HDMACs contained flattened cells, not ovary-shapedchondrocyte-like cells (Figure 2). Combined with thefact that the HDMACs were not stained with safranin O(Figure 1d), these cells are suggested to be undifferentiated. On the other hand, we also confirmed that the constructs could undergo differentiation into chondrocytes
in vitro when cultured in chondrocyte induction medium(data not shown). In vivo, the implanted MSCs spontaneously differentiate into articular cartilage and subchondral bone; eventually, the normal structures of bone
and cartilage were completely formed (Figures 6 and 7).
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implanted area. This suggests that hypertrophy of articular cartilage, one of the major problems using MSC-based
constructs, did not occur during the regeneration period.
Our study is distinguished not only by the use ofundifferentiated MSCs but also by the use of a constructgenerated by a scaffold-free method. Our HDMACs haveenough volume to fill a full-thickness cartilage defect.
Cell nutrition particularly in the middle of a big construct can be a concern [24]. However, the nucleus was
stained with hematoxylin in the middle of the HDMACs
and there was no fragmentation or chromatin condensation (Figure 1d). This suggests that the cells in the construct were viable. Previous studies developed various
scaffold-free methods using chondrocytes or MSCs; thesimplest reported method is injection of synovial MSCs
into a cartilage defect in vivo [16]. In that study, however, although the histological score was significantly
greater than the control, the depth of the defect was restricted to 2 mm and the surface was concave. Anothermethod using a scaffold-free construct derived from synovial MSCs resulted in successful repair of a cartilage
defect [14], but again, the depth was limited to 2 mm. In
yet another study, scaffold-free chondrocyte sheets wereused successfully to repair of a partial-thickness cartilage
defect in vivo [13]. However, this method was insufficient for subchondral bone repair and tissue integrationin the context of a full-thickness cartilage defect [25]. In
efforts to obtain enough cell mass to fill a full-thickness
cartilage defect, pellet cultures of chondrocytes or MSCs
have been investigated. In one animal study, chondrocyte
pellet implantation into a 3-mm osteochondral defect was
attempted [15]. Although the thickness of the repaired
cartilage remained relatively constant, overgrowth of thesurface was observed, and subchondral bone regeneration
was extremely limited.

Acknowledgements
The authors thank Dr. Ken Okazaki, Department of Orthopaedic Surgery at
Kyushu University Hospital, for the support in preparing the manuscript andDr. Takaaki Kanemaru, Morphology Core Unit at Kyushu University, for kindly
observing the HDMACs by SEM. This study was partially supported by JST
Translational Research Network Program No. 08061012.

Conclusions
Our novel technique, using a three-dimensional scaffoldfree autologous construct derived from BM-MSCs, successfully achieved the simultaneous regeneration of bone
and cartilage for up to 1 year in vivo. This method has
potential for clinical use as a safe and effective method
for repairing bone and cartilage defects.
Competing interests
KN is a co-founder and a shareholder of Cyfuse Biomedical K.K., Tokyo, Japan.
The other authors declare that they have no competing interests.

Authors’ contributions
KN designed the study and executed the experimental setup. KI participatedin the data analysis and drafted the manuscript. SA participated in theexecution of the experiment and discussion. SM and YI provided important
feedback on the experimental setup and participated in the discussion.
All authors read and approved the final version of the manuscript.

Received: 27 June 2014 Accepted: 7 October 2014

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Cite this article as: Ishihara et al.: Simultaneous regeneration of
full-thickness cartilage and subchondral bone defects in vivo using athree-dimensional scaffold-free autologous construct derived fromhigh-density bone marrow-derived mesenchymal stem cells. Journal of
Orthopaedic Surgery and Research 2014 9:98.

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