A Pre-market, Multi-center, International, Open-label, Single-arm Study to Evaluate the Safety and Performance of a Class III Medical Device (GreenBone Implant) for Surgical Repair of Long Bone Defects
Phase: NA
Conditions: Bone Substitutes, Bone Defect
Sponsor: GreenBone Ortho S.p.A.

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
Study Synopsis  
 
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STUDY SYNOPSIS 
 
 
Study Title 
A pre-market, multi-center, international, open-label, single-arm study to evaluate 
the safety and performance of a Class III medical device (GreenBone Implant) for 
surgical repair of long bone defects 
Study Code 
GB-02-18 
 
Study Sponsor
GreenBone Ortho S.r.l. 
Via Albert Einstein, 8 
48018 Faenza (RA) – Italy 
Clinical Phase Pre-market clinical investigation of a Class III Investigational Medical Device. 
 
 
 
 
 
 
 
 
Study Design 
 
Multi-center, international, prospective, open-label, single-arm, first-in-human 
clinical investigation. The Patients enrolled in this clinical investigation will 
undergo a scheduled surgery for the treatment of long bone defects up to 6 cm using 
GreenBone Implant. After the surgery, the Patients will be monitored at pre-
scheduled visits up to 12 months. Adverse events, pain, quality of life and 
functional parameters, as well as X-ray and CT scan, will be evaluated at scheduled 
follow-up visits. An independent DSMB will review the safety reports at regular 
intervals and Serious Adverse Events (SAE) as soon as reported, to protect Patients 
participating in the study. The initial phase of the study contemplates the treatment 
of bone defects up to 3 cm. An adaptive interim analysis will be performed when 
the first 7 Patients will have completed the 6-month follow-up visit. The DSMB 
will review the results of the interim analysis with respect to the primary endpoint 
(safety), and provide one of the following recommendations to the Sponsor: a) to 
stop the study for unacceptable frequency and severity of adverse events or b) to 
continue the study up to 25 Patients recruited and to include at least 5 Patients with 
a longer bone defect (> 3 cm up to 6 cm). 
Investigational 
Medical 
Device 
GreenBone Implant for bone substitution, a Class III Medical Device. The 
GreenBone Implant had not yet received the CE mark at the time of the Study first 
submissions and first patient enrollment. The Device received the CE mark in 
December 2019.  
Investigational 
Medical 
Device Use 
 
GreenBone  Implant is used in  the  surgical  procedures  for repair of bones 
defects. 
 
Investigational 
Medical 
Device 
Description 
GreenBone Implant is a ceramic reabsorbable scaffold with a geometrical structure, 
engineered to reflect anatomical and physiological bone hierarchical structures. 
GreenBone Implant scaffold is made of biomimetic substituted calcium phosphate 
phases (HA and β-TCP and minerals). It is suitable for repair of defects in bones. 
GreenBone Implant is a EU class III Medical Device. 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
Study Synopsis  
 
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Clinical 
Investigation 
Rationale 
Bone defects can result from malformation, high-energy traumatic events, bone 
resection due to different pathologies such as tumors or infections, or from the 
treatment of complex non-unions, all very challenging conditions in orthopedic 
practice. 
 
Current treatment of bone defects usually implies the use of bone grafts and/or 
biocompatible materials, to create a scaffold that bridges the defect, favoring the 
migration of cells from the neighboring tissues to fill the gap. Autograft and 
Allograft are the treatments most currently used for large bone loss, but both 
treatments have significant disadvantages. Different types of synthetic materials 
have been tested for repair of long bone gaps. 
 
However, at the moment, there is not any bone substitute with the ideal/desirable 
osteoinductive, osteoconductive and mechanical properties. GreenBone Implant is 
a synthetic, acellular, reabsorbable, new generation bone graft, being suitable for 
surgical reconstruction of bone defects. 
 
 
 
 
 
 
Study 
Objectives 
The primary objective of this study is to assess the safety profile of the GreenBone 
Implant in Patients undergoing surgery for the treatment of bone defects up to 6 
cm, specifically for long bone repair. The initial phase of the study contemplates 
the treatment of bone defects up to 3 cm. Following DSMB interim assessment, at 
least 5 Patients, of the 25 patients, with a longer bone defect (> 3 up to 6 cm) will 
be enrolled. 
 
The secondary objectives of this study are: 
- 
To evaluate the progression of bone healing and bone regeneration at the 
level of the intervention site after use of GreenBone Implant throughout a 
12-month observation period; 
- 
To evaluate the degree of pain throughout a 12-month observation period; 
- 
To evaluate the functional recovery of Patient throughout a 12-month 
observation period. 
- 
To evaluate the health related quality of life of Patient throughout a 12- 
month observation period. 
Implant of the 
Investigational 
Medical 
Device 
GreenBone Implant is applied to the bone gap by the surgeon during the scheduled 
intervention, according to standard clinical practice and to the information 
provided by GreenBone Ortho S.r.l. in the device Instructions For Use (IFU) 
document. 
 
 
Study 
Population 
Male and female Patients, aged between 18 and 65 years, undergoing surgery for 
the treatment of long bone defects up to 6 cm. 
 
Please note: The initial phase of the study contemplates the treatment of bone 
defects up to 3 cm. After an adaptive interim analysis review by the DSMB, 
inclusion criteria will be amended; specifically to include the recruitment of 
patients  suffering from bone defects up to 6 cm. 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
Study Synopsis  
 
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Study 
Sample Size 
 
In the absence of published guidance on first-in-human trials on long bone surgery, 
the sample size (n=25 including a 20% drop-out rate) has been chosen to be 
consistent with first-in-human studies of pharmacological agents which are usually 
conducted in 10-15 patients (Rubinstein LV et al 2010). 
 
 
 
 
 
 
 
 
 
Inclusion 
Criteria 
1. Male or female Patients. 
2. Patients aged ≥ 18 and ≤ 65 years old. 
3. Patients requiring long bone defect reconstructions up to 3 cm, resulting 
from high-energy traumatic events, or from the treatment of complex bone 
non-unions in the extremities (arms and legs), or patients undergoing the 
second stage of the Masquelet technique. 
4. Patients understanding the nature of the study and providing their informed 
consent to participation. 
5. Patients willing and able to attend the follow-up visits and procedures 
foreseen by study protocol. 
 
Please note: after interim analysis review by the DSMB, inclusion criteria n. 3 will 
be modified as follows (all Investigators will be notified by the Sponsor, and 
adequate training by CRA will be conducted): 
 
Change of inclusion criteria n. 3: Patients requiring long bone defect 
reconstructions up to 6 cm, resulting from high-energy traumatic events, or from 
the treatment of complex bone non-unions in the extremities (arms and legs), or 
patients undergoing the second stage of the Masquelet technique. 
 
 
 
 
 
 
 
 
 
Exclusion 
Criteria 
For the specific characteristics of this study, the final decision on Patient enrolment 
will be made by the Investigator at time of surgery (during surgery, before 
implantation). 
 
1. Patients with bone infection at the time of enrolment. 
2. Patients with bone malignant tumor(s) at the time of enrolment. 
3. Patients who have been treated with chemotherapy or radiotherapy within 
12 months before the enrolment. 
4. Patients with concomitant infectious systemic diseases at the time of 
enrolment. 
5. Patients with known inflammatory systemic diseases at the time of 
enrolment. 
6. Patients with concomitant myeloproliferative disorders at the time of 
enrolment. 
7. Patients currently treated with systemic immunosuppressive agents, 
excluding steroids. 
8. Patients with active autoimmune disease. 
9. Patients 
with 
a 
pre-existing 
calcium 
metabolism 
disorder 
(i.e. hypercalcemia). 
10. Known hyperthyroidism or autonomous thyroid adenoma. 
11. Patients with coagulopathy or bleeding disorders. 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
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12. Patients who are not allowed to undergo the study procedures involving 
imaging (X-rays, CT scan) based on Investigator’s judgement. 
13. Patients with known or suspected allergy or hypersensitivity to the 
GreenBone Implant components. 
14. Patients who are participating or have participated in other clinical studies 
within the 30 days before the study enrolment. 
15. Patients identified by the Investigator to have intra-operative findings that 
may preclude conduct of study procedure. 
16. Patients with occurrence of major intra-operative complications that require 
resuscitation or deviation from the planned surgical procedure. 
17. Women who are pregnant or breast-feeding or who wish to become 
pregnant during the period of the clinical investigation and for three months 
later. 
18. Female Patients of childbearing age (less than 24 months after the last 
menstrual cycle) who do not use adequate contraception *. 
 
* Methods at low risk of contraceptive failure (less than 1% per year) when used 
consistently, including: combined (estrogen and progestogen containing) 
hormonal contraception associated with inhibition of ovulation (oral, intravaginal, 
transdermal), progestogen-only hormonal contraception associated with 
inhibition of ovulation (oral, injectable, implantable), some intra-uterine devices. 
 
Please note: exclusion criteria after interim analysis will remain the same. 
Not Allowed 
Concomitant 
Medications 
 
Systemic immunosuppressive agents, excluding steroids. 
 
 
 
 
 
 
 
 
Study Flow 
Chart 
Visit 
Procedures 
Day 
 
 
 
 
 
 
 
 
Visit 1 
(V1 – Screening) 
 Eligibility criteria evaluation 
 Demography / medical history 
 Informed consent collection 
 Patient enrolment 
 Standard preoperative lab assessment 
 Vital signs 
 Physical examination 
 ECG 
 X-Ray examination 
 CT scan examination (if necessary) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Pregnancy test (for female Patients of 
childbearing age) 
 
 
 
 
 
 
 
 
Up to 7 
days before 
V2 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
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Visit 2 
(V2 – Surgery) 
Before surgery: 
 Informed Consent confirmation by the 
Patient 
During and after surgery: 
 Device implantation: application of  the 
GreenBone Implant at the target site 
 Evaluation of technical success 
 Concomitant medications 
 Adverse events 
 Provide Implant Card to each patient 
 Return of discarded devices (if any) and 
completion 
of 
the 
report 
of 
Investigational       Medical       Device 
Deficiency 
 
 
 
 
 
 
 
D 0 
 
 
 
 
 
Visit 3 
(V3 – Control 1) 
 Physical examination of the operated 
limb 
 X-Ray examination 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +30 ±3 
(1M) 
 
 
 
 
 
Visit 4 
(V4 – Control 2) 
 Physical examination of the operated 
limb 
 X-Ray examination 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +60 ±5 
(2M) 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
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Visit 5 
(V5 – Control 3) 
 Physical examination of the operated 
limb 
 X-Ray examination 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +90 ±7 
(3M) 
 
 
 
 
 
 
Visit 6 
(V6 – Control 4) 
 X-Ray examination 
 CT scan examination (if healing 
activity inconclusive on radiographs) 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +180 ±14 
(6M) 
 
 
 
 
 
Visit 7 
(V7 – Control 5) 
 X-Ray examination 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +270 ±14 
(9M) 
 
 
 
 
 
Visit 8 
(V8 – Control 6) 
 X-Ray examination 
 CT scan examination (if healing 
activity inconclusive on radiographs) 
 Evaluation of global tolerability of 
Patient (by the Investigator) 
 Quality of Life (EuroQoL EQ-5D-5L 
Validated Questionnaire) 
 VAS score (0-10 point scale) 
 Functional assessment (36-Item Short 
Form Survey – SF-36) 
 Concomitant medications 
 Adverse events 
 
 
 
 
 
D +360 ±14 
(12M) 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
Study Synopsis  
 
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Should any of the Patients request at any time an additional visit (i.e. in case of 
discomfort in the area of intervention), an additional visit should be organized to 
allow examination by the Investigator. 
 
 
 
Study 
Endpoints 
Primary Endpoint: 
 
Evaluation of adverse events rate during the study period. All adverse occurrences 
(serious/non-serious or device-related/non device-related) will be recorded 
prospectively, categorized and evaluated for causality using defined criteria. 
 
The study will be monitored by an independent Data Safety Monitoring Board 
(DSMB). 
 
 
 
 
 
 
 
 
 
 
 
Study 
Endpoints 
Secondary Endpoints: 
 
1. Evaluation of technical success, defined as successful delivery of the 
GreenBone Implant in the target defect bone evaluated at V2 (during 
surgical procedure). 
2. Evaluation of the bone regeneration in the area of intervention in 
comparison to baseline (V1), by means of X-ray examination, on visits V3, 
V4, V5, V6, V7 and V8. X-ray scoring systems have been implemented 
according to the criteria proposed by Lane and Sandhu (bone formation, 
union and remodeling) (Lane 1987) (See Appendix 1). 
3. Evaluation of the bone regeneration in the area of intervention in 
comparison to baseline (V1), by means of CT scan examination on visits 
V6 and V8 if indicated. 
4. Functional Assessment: evaluation of functional recovery of Patient, by 
means of a standardized questionnaire (36-Item Short Form Survey – SF- 
36), on visits V1, V3, V4, V5, V6, V7 and V8 (See Appendix 2). 
5. Evaluation of degree of pain perceived by the Patient, by means of a VAS 
(0-10 point scale), on visits V1, V3, V4, V5, V6, V7 and V8 (Price 1983) 
(See Appendix 3). 
6. Evaluation of Quality-of-Life by means of EuroQoL EQ-5D-5L Validated 
Questionnaire, on visits V1, V3, V4, V5, V6, V7 and V8 (See Appendix 4).
7. Evaluation of global tolerability to treatment of Patient (by the 
Investigator), through a standardized questionnaire, on visits V3, V4, V5, 
V6, V7 and V8 (See Appendix 5). 
8. Evaluation of the number of re-interventions occurring for the Patient 
throughout the 12-month observation period. 

Study Protocol No: GB-02-18                        CONFIDENTIAL                              Ver. 3.0 dated 04/03/2020                       
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Statistical 
Analysis 
 
A descriptive analysis of safety and performance variables will be performed. 
 
The descriptive analyses will be performed in order to describe the nature of 
adverse events, their distribution and frequency. Moreover, all factors which may 
appear predictive for the rate of adverse events will be analyzed (age, sex, 
dimension of the implanted device, system used, diabetes history, smoking habits, 
alcohol consumption, concomitant treatments/diseases, other). If the percentage 
of Patients with device-related AE would be sufficiently large in respect to the 
overall sample (≥ 70%) to allow an evaluation of the time interval between the date 
of surgery and the date of the first adverse event occurence, a median of the time 
interval will be estimated, with 95% Confidence Interval, through the Kaplan-
Meier survival analysis. 
 
An adaptive interim analysis will be performed when the first 7 Patients will have 
completed the 6-month follow-up visit. An independent Data Safety Monitoring 
Board (DSMB) will review the results of the interim analysis with respect to the 
primary endpoint (safety), and provide one of the following recommendations to 
the Sponsor: a) to stop the study for unacceptable frequency and severity of adverse 
events or b) to continue the study up to a minimum of 25 Patients recruited and to 
include at least 5 Patients with a longer bone defect (> 3 up to 6 cm). 
 
The independent DSMB will receive all the safety data at regular intervals and the 
data about Serious Adverse Events (SAE) as soon as reported, to protect Patients 
participating in the study. 
Investigational 
Sites 
The study will involve 8 investigational sites in Bosnia & Herzegovina, Israel, 
Italy, Serbia, Slovenia, Switzerland and UK.