Engineered phalangeal grafts for children with symbrachydactyly: A proof of concept.

Tissue engineering approaches hold great promise in the field of regenerative medicine, especially in the context of pediatric applications, where ideal grafts need to restore the function of the targeted tissue and consider growth. In the present study, we aimed to develop a protocol to engineer autologous phalangeal grafts of relevant size for children suffering from symbrachydactyly. This condition results in hands with short fingers and missing bones. A previously-described, developmentally-inspired strategy based on endochondral ossification (ECO)-the main pathway leading to bone and bone marrow development-and adipose derived-stromal cells (ASCs) as the source of chondroprogenitor was used. First, we demonstrated that pediatric ASCs associated with collagen sponges can generate hypertrophic cartilage tissues (HCTs) in vitro that remodel into bone tissue in vivo via ECO. Second, we developed and optimized an in vitro protocol to generate HCTs in the shape of small phalangeal bones (108-390 mm3) using freshly isolated adult cells from the stromal vascular fraction (SVF) of adipose tissue, associated with two commercially available large collagen scaffolds (Zimmer Plug® and Optimaix 3D®). We showed that after 12 weeks of in vivo implantation in an immunocompromised mouse model such upscaled grafts remodeled into bone organs (including bone marrow tissues) retaining the defined shape and size. Finally, we replicated similar outcome (albeit with a slight reduction in cartilage and bone formation) by using minimally expanded pediatric ASCs (3 × 106 cells per grafts) in the same in vitro and in vivo settings, thereby validating the compatibility of our pediatric phalanx engineering strategy with a clinically relevant scenario. Taken together, these results represent a proof of concept of an autologous approach to generate osteogenic phalangeal grafts of pertinent clinical size, using ASCs in children born with symbrachydactyly, despite a limited amount of tissue available from pediatric patients.

Pull-out vs. suture in zone Ia-Ib flexor tendon injuries: clinical results from a multicentre cohort study.

BACKGROUND: Several surgical techniques have been reported for flexor tendon zone 1a-b lacerations without a clear consensus on the gold standard treatment. The purpose of this multicentre study was to measure the outcomes of zone 1a-b flexor tendon injuries treated with a pull-out suture (POS) versus direct suture (DS) technique. PATIENTS AND METHODS: Fifteen patients were treated with the pull-out technique and 22 patients with a direct suture technique between 2014 and 2020. The controlled active motion (CAM) regimen protocol and a standardised follow-up schedule were used in both groups. Data on the demographics, surgery, and treatment characteristics were collected at baseline as well as at week 6 and 13 post-operatively. The primary outcome measurement was the complication rate. Secondary outcome measurements were reoperation rate, finger range of motion (ROM), strength as well as patient satisfaction. RESULTS: The patient age ranged from 18 to 75 years in both groups with all patients having a complete FDP lesion after a clean-cut injury. The complication rate was 41% in the POS group and 16% in the DS group. The reoperation rate was 29% in the POS group and 8% in the DS group. Significant between-group differences in favour of the DS group were found in the passive DIP and passive PIP+DIP ROM at week 6 and week 13. Mean hand strength was 28.7 (11.6) kg in the POS group at week 13 and 21.3 (7.9) kg in the DS group. This difference was statistically significant (p=0.012) with a medium effect size (r=-0.41). Average patient satisfaction at week 13 was 7.5 (2.2) points in the POS and 7.7 (1.3) points in the DS group. The difference between groups was not significant (p=0.839). CONCLUSION: This register-based study demonstrates lower complication and reoperation rates with the direct suture approach compared with a pull-out button technique. Although clinical results were similar between the two surgery techniques at week 13 post-surgery, a direct suture approach should always be attempted whenever possible. If necessary, other local structures should be included to increase suture strength and allow for early active motion rehabilitation regimens.

Relationship between pain, nerve injury and clinical outcomes after flexor tendon injuries in zones 1-2: a retrospective cohort study.

Introduction: The influence of pain and a concomitant digital nerve injury on the course of rehabilitation after flexor tendon injury remains ambiguous. The objectives of the study were to: i) analyse the evolution of pain spanning one year after a primary flexor tendon repair in zones 1-3; ii) examine the differences in pain levels in patients with and without digital nerve injury; and iii) evaluate the relationship between pain, digital nerve injury and pain medication, total active motion (TAM), DASH scores and patient satisfaction. Methods: Data from 189 patients were retrieved from a flexor tendon registry between 2014 and 2020. Differences in pain, TAM, DASH and patient satisfaction were analysed. Multiple linear and binary logistic regression analyses were performed to determine the relationship between clinical outcomes. Results: Pain significantly decreased in the course of rehabilitation (p < 0.001 to 0.006). No relationship could be identified between nerve injury and pain (p = 0.21-0.97). In week 6, the presence of pain and a nerve injury were significantly associated with lower TAM scores (p = 0.001). In week 13, pain during motion (p < 0.001) and the presence of a nerve injury (p = 0.036) were significantly associated with worse DASH scores. Patient satisfaction was significantly inversely correlated to pain during motion in weeks 13 and 26 (p < 0.001). Conclusion: We found a significant relationship between pain during motion and pain medication intake, TAM, DASH scores and patient satisfaction. It is therefore advisable to closely monitor this parameter after flexor tendon injuries. Study registration: This multi-center cohort study is registered under https://clinicaltrials.gov: NCT04312412.

Vascularized Growth Plate Transfer in Paediatric Ulna Non-Union: Operative Technique and Review of the Literature.

Congenital pseudarthrosis of forearm fractures is rare and is strongly associated with neurofibromatosis type 1 (NF1). Our case report illustrates the progression of a non-union of the ulna after minor trauma in a twelve-year-old boy, newly diagnosed with NF1, and presents the technique of microsurgical bone reconstruction, including the growth plate. More than seven years after the first operation, follow-up presents a favorable outcome with a pain-free patient and unrestricted function of the forearm after a secondary correction of the remaining radial bowing. This treatment is discussed with a comprehensive review of the current literature on ulnar congenital pseudarthrosis in PubMed and Google Scholar and free fibular growth plate transfer in PubMed and Google Scholar. Nine publications reporting on 20 cases of congenital ulnar non-unions were identified. With this reconstructive option, favorable outcomes were achieved in all cases with the union after primary surgery and complications requiring further surgeries in nine cases. The benefit of vascularized growth plate bone transfer in congenital ulna non-union seems to be significant compared to other therapies such as open reduction internal fixation (ORIF), non-vascularized bone grafts, or one-bone-forearms and beneficial when growth reconstruction is needed. Other techniques might be necessary to improve insufficient long-term results.

Conservative and Surgical Treatment of Osteochondromas in Children, Particularly with or without Surgical Lengthening of the Ulna.

Prevention of rotatory impairment and radial head dislocation in the forearm is an important aspect when treating children with osteochondromas. Various studies tried to determine the best treatment, describing different surgical techniques. No consensus has been reached yet. This retrospective study compares the treatment outcome of patients with osteochondroma of the radius and ulna after surgical or conservative treatment. Seventeen forearms treated over a period of 20 years were analysed. Outcome parameters were the prospectively collected clinical data and the radiological findings: "relative shortening" of ulna/radius, the "radial articular angle" (RAA) and the "carpal slip" (CS). Our study shows an improvement of the range of motion and cosmetic appearance of the forearm after an operative procedure, with or without bone lengthening. We observed an increase in wrist and elbow mobility with a decrease in pain scores and a confirmed high cosmetic satisfaction in almost 70% of the patients after bone lengthening and up to 85% after simple excision. For patients suffering from functional impairment or pain, an operative approach is beneficial. Multiple and repetitive osteochondroma excisions are recommended during growth to prevent deformity and rotatory motion restriction. Lengthening procedures require a careful indication.

Early Postoperative Recovery after Modified Ultra-Minimally Invasive Sonography-Guided Thread Carpal Tunnel Release.

Thread carpal tunnel release (TCTR) has been reported to be safe and effective for the treatment of carpal tunnel syndrome. The aim of this study is to evaluate the modified TCTR for safety, efficacy, and postoperative recovery. Seventy-six extremities in 67 patients undergoing TCTR were analyzed pre- and postoperatively using clinical parameters and patient-reported outcome measures. Twenty-nine men and 38 women with a mean age of 59.9 ± 18.9 years underwent TCTR. The mean postoperative time to resume activities of daily living was 5.5 ± 5.5 days, analgesia was completed after 3.7 ± 4.6 days, and return to work was achieved after a mean of 32.6 ± 15.6 days for blue-collar workers and 4.6 ± 4.3 days for white-collar workers. The Boston Carpal Tunnel Questionnaire (BCTQ) and Disability of Arm, Shoulder, and Hand (DASH) scores were comparable with previous studies. Overall, two persistent compressions and one recurrence required open reoperation (3.9%). All three had been operated in the initial phase, and none required reoperation after an additional safety step was introduced. No other complications occurred. TCTR surgery appears to be a safe and reliable technique with almost no wound and scarring and a potentially faster recovery time than open techniques. Although our technical modifications may reduce the risk of incomplete release, TCTR requires both ultrasound and surgical skills and has a considerable learning curve.

Six-Strand Flexor Pollicis Longus Tendon Repairs With and Without Circumferential Sutures: A Multicenter Study.

BACKGROUND: The purpose of this study is to assess outcomes in flexor pollicis longus tendon repairs with 6-strand core sutures with and without circumferential sutures. METHODS: A 6-strand core suture technique with and without circumferential sutures was used. Thirty-three patients were summarized in the C group (circumferential group) and 16 patients in the NC group (non-circumferential group). After the surgery, the wrist was stabilized with a dorsal blocking splint and a controlled early active motion protocol was applied. At weeks 6, 13, and 26 data on demographics, type of injury, surgery, postoperative rehabilitation, complications such as re-rupture and the following outcome measurements were collected: range of motion and its recovery according to the Tang criteria, Kapandji score, thumb and hand strengths, Disabilities of the Arm, Shoulder and Hand score, and satisfaction. RESULTS: There were no significant differences in range of motion and strength between the 2 treatment groups. In both groups, the outcome measurements increased over time and they expressed similar satisfaction with the surgical treatment. In 4 patients of the C group tendon repair ruptured and in 1 patient of the NC group. CONCLUSIONS: Six-strand repair technique is an effective procedure to assure early active motion after flexor pollicis longus tendon injuries and good results can also be achieved by omitting the circumferential suture.

Chronic Recurrent Multifocal Osteomyelitis in a Child's Hand: Case Report and Review.

We present a 9-year-old girl with persistent pain and swelling of the left wrist. X-ray, magnetic resonance imaging (MRI) and bone biopsy led to the diagnosis of chronic recurrent multifocal osteomyelitis (CRMO), affecting phalangeal and metacarpal bases and distal carpal bones on the ulnar side of the wrist. She was treated with non-steroidal anti-inflammatory drugs and complete remission with no long-term sequelae was achieved. CRMO is a rare auto-inflammatory condition with infrequent involvement of the hand. The current literature is discussed. The aim of this report is to raise awareness of the condition to reduce the time to diagnosis and unnecessary antibiotic treatment and to prevent permanent disability due to the progression of the disease. Level of Evidence: Level V (Therapeutic).

Wrist Arthroscopy Is Effective for the Diagnosis and Treatment of Chronic Wrist Pain in Pediatric Patients.

Purpose: To determine the outcomes of diagnostic and therapeutic arthroscopy in patients with chronic wrist pain who are younger than 16 years of age. Methods: We retrospectively analyzed collected data from medical records of patients who had undergone wrist arthroscopy by the senior author between 2015 and 2017 for longstanding wrist pain and were 16 years old or younger. Findings from preoperative magnetic resonance imaging (MRI) were compared with the intraoperative diagnosis, and midterm results were gathered by a telephone interview. Results: Ten patients were included. Eight of 10 patients had undergone conservative therapy before surgery, and 60% had a history of single trauma. In 6 of 10 patients, the arthroscopic diagnosis correlated with the MRI findings. Eight of 10 wrists (80%) showed a tear of the triangular fibrocartilage complex (TFCC). Only 4 of the 8 TFCC tears were correctly identified by 3-Tesla MRI. Conclusions: In our study, wrist arthroscopy was an effective tool not only to diagnose but also to treat relevant TFCC lesions both in adolescents and children suffering from persistent wrist pain. A 3-Tesla MRI was neither sensitive nor specific enough to correctly diagnose lesions in small pediatric wrists. Level of Evidence: IV, Therapeutic case series.

Cross-sectional Vascularization Pattern of the Adipofascial Anterolateral Thigh Flap for Application in Tissue-engineered Bone Grafts.

As part of the engineering of bone grafts, wrapping constructs in well-vascularized tissue, such as fascial flaps, improves bone formation. Our aim was to understand the cross-sectional vascularization pattern of human adipofascial flaps for this application. METHODS: Seven adipofascial anterolateral thigh (ALT) flaps were harvested from five human cadaveric specimens. Axial vessel density was analyzed by immunohistochemistry and quantitative histology. RESULTS: We found a high density of blood vessels directly superficial to and close to the fascia. A secondary plexus in between this first suprafascial plexus and the subdermal plexus was also identified. In all specimens, this second plexus showed less vascular density, and appeared to be at a constant level within the suprafascial fat throughout the flaps. The peak measurements for this secondary plexus varied between 1.2 and 2 mm above the deep fascia, depending on the donor's body mass index. CONCLUSIONS: Quantitative immunohistochemistry is a reliable method to quantify and locate vessel density in an adipofascial flap. This is vital information before wrapping nonvascularized material into such a flap to estimate the inosculation potential of these vessels and likelihood of survival of the tissue. To profit from both suprafascial vascular plexuses, a correlation between subcutaneous tissue thickness and distance of the second plexus to the fascia should be further investigated. For the moment, we recommend maintaining at least 2-3 mm of subcutaneous fatty tissue on the fascia, to profit from both plexuses. Engineered constructs should be wrapped on the superficial medial side of the fascial flap to enhance vascularization.

3D-Printed Hand Splints versus Thermoplastic Splints: A Randomized Controlled Pilot Feasibility Trial.

In this randomized controlled pilot trial, we compared three-dimensional (3D)-printed made-to-measure splints to conventional custom-made thermoplastic splints. In a clinical setting, we evaluated their general applicability and possible benefits for immobilization in hand surgical patients. We included 20 patients with an indication for immobilization of at least 4 weeks, regardless of the splint design. Patient comfort and satisfaction were assessed with questionnaires at splint fitting, as well as 2 and 4-6 weeks later. The 3D splints were designed and printed in-house with polylactic acid from a 3D surface scan. Our data suggest that 3D-printed splinting is feasible, and patient satisfaction ratings were similar for 3D-printed and thermoplastic splints. The 3D splint production process needs to be optimized and other materials need to be tested before routine implementation is possible or more patients can be enrolled in further studies. Validated quality assessment tools for current splinting are lacking, and further investigation is necessary.

Case Report: Reconstruction of a Large Maxillary Defect With an Engineered, Vascularized, Prefabricated Bone Graft.

The reconstruction of complex midface defects is a challenging clinical scenario considering the high anatomical, functional, and aesthetic requirements. In this study, we proposed a surgical treatment to achieve improved oral rehabilitation and anatomical and functional reconstruction of a complex defect of the maxilla with a vascularized, engineered composite graft. The patient was a 39-year-old female, postoperative after left hemimaxillectomy for ameloblastic carcinoma in 2010 and tumor-free at the 5-year oncological follow-up. The left hemimaxillary defect was restored in a two-step approach. First, a composite graft was ectopically engineered using autologous stromal vascular fraction (SVF) cells seeded on an allogenic devitalized bone matrix. The resulting construct was further loaded with bone morphogenic protein-2 (BMP-2), wrapped within the latissimus dorsi muscle, and pedicled with an arteriovenous (AV) bundle. Subsequently, the prefabricated graft was orthotopically transferred into the defect site and revascularized through microvascular surgical techniques. The prefabricated graft contained vascularized bone tissue embedded within muscular tissue. Despite unexpected resorption, its orthotopic transfer enabled restoration of the orbital floor, separation of the oral and nasal cavities, and midface symmetry and allowed the patient to return to normal diet as well as to restore normal speech and swallowing function. These results remained stable for the entire follow-up period of 2 years. This clinical case demonstrates the safety and the feasibility of composite graft engineering for the treatment of complex maxillary defects. As compared to the current gold standard of autologous tissue transfer, this patient's benefits included decreased donor site morbidity and improved oral rehabilitation. Bone resorption of the construct at the ectopic prefabrication site still needs to be further addressed to preserve the designed graft size and shape.

Correction of Thumb Duplication: A Systematic Review of Surgical Techniques.

Surgical intervention for thumb duplication can be divided into three categories: simple excision of the accessory thumb, excision of the accessory thumb with reconstruction from available "spare parts," and combining the two thumbs into one, as described by Bilhaut. This prospectively PROSPERO registered systematic review evaluates the overall, aesthetic and functional outcomes for the latter two options (reconstruction from spare parts vs. combining two thumbs into one), aiming to facilitate evidence-based decision making when addressing thumb duplication and direct future research. The review was performed in accordance with the Cochrane Handbook of Systematic Reviews and PRISMA statement. Embase, PubMed, Medline, and Cochrane databases were systematically searched. Studies offering comparisons of techniques were included. Risk of bias was assessed using the Risk of Bias In Non-randomized Studies-of Intervention tool. The quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation. Ten retrospective observational studies were included. Data did not consistently allow analysis by procedure type. Four studies reported similar overall outcomes between techniques, while two specifically reported poor overall outcomes for the Bilhaut procedure. Two studies reported comparatively worse aesthetic outcomes for the Bilhaut procedure with four studies reporting comparatively improved functional outcomes for this procedure. Overall, interpretation of outcomes was challenging with no patient-reported outcome measures used. The quality of the evidence was universally "very low" due to all studies being at risk of methodological bias. Based on the available evidence, surgical techniques for thumb duplication correction appear comparable regarding overall outcome. There is limited evidence suggesting reconstruction with spare parts offers superior aesthetic outcomes at the expense of stability. The level of evidence is III.

VEGF Over-Expression by Engineered BMSC Accelerates Functional Perfusion, Improving Tissue Density and In-Growth in Clinical-Size Osteogenic Grafts.

The first choice for reconstruction of clinical-size bone defects consists of autologous bone flaps, which often lack the required mechanical strength and cause significant donor-site morbidity. We have previously developed biological substitutes in a rabbit model by combining bone tissue engineering and flap pre-fabrication. However, spontaneous vascularization was insufficient to ensure progenitor survival in the core of the constructs. Here, we hypothesized that increased angiogenic stimulation within constructs by exogenous VEGF can significantly accelerate early vascularization and tissue in-growth. Bone marrow stromal cells from NZW rabbits (rBMSC) were transduced with a retroviral vector to express rabbit VEGF linked to a truncated version of rabbit CD4 as a cell-surface marker. Autologous cells were seeded in clinical-size 5.5 cm3 HA scaffolds wrapped in a panniculus carnosus flap to provide an ample vascular supply, and implanted ectopically. Constructs seeded with VEGF-expressing rBMSC showed significantly increased progenitor survivival, depth of tissue ingrowth and amount of mineralized tissue. Contrast-enhanced MRI after 1 week in vivo showed significantly improved tissue perfusion in the inner layer of the grafts compared to controls. Interestingly, grafts containing VEGF-expressing rBMSC displayed a hierarchically organized functional vascular tree, composed of dense capillary networks in the inner layers connected to large-caliber feeding vessels entering the constructs at the periphery. These data constitute proof of principle that providing sustained VEGF signaling, independently of cells experiencing hypoxia, is effective to drive rapid vascularization and increase early perfusion in clinical-size osteogenic grafts, leading to improved tissue formation deeper in the constructs.

Tissue engineering for paediatric patients.

The effects of oncological treatment, congenital anomalies, traumatic injuries and post-infection damage critically require sufficient amounts of tissue for structural and functional surgical reconstructions. The patient’s own body is typically the gold standard source of transplant material, but in children autologous tissue is available only in small quantities and with severe morbidity at donor sites. Engineering of tissue grafts starting from a small amount of autologous material, combined with suitable surgical manipulation of the recipient site, is expected to enhance child and adolescent health, and to offer functional restoration for long-term wellbeing. Moreover, engineered tissues based on patient-derived cells represent invaluable models to investigate mechanisms of disease and to develop/test novel therapeutic approaches. In view of these great opportunities, here we introduce the currently limited successful implementation of tissue engineering in paediatric settings and discuss the open challenges in the field. A particular focus is on the specific needs and envisioned strategies in the areas of bone and osteochondral regeneration in children.

Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid.

Objectives: Bone ischemia and necrosis are challenging to treat, requiring investigation of native and engineered bone revascularisation processes through advanced imaging techniques. This study demonstrates an experimental two-step method for precise bone and vessel analysis in native bones or vascularised bone grafts using X-ray microtomography (µCT), without interfering with further histological processing. Methods: Distally ligated epigastric arteries or veins of 6 nude rats were inserted in central channels of porous hydroxyapatite cylinders and these pedicled grafts were implanted subcutaneously. One week later, the rats were perfused with ink-gelatin and euthanised and the femurs, tibias, and grafts were explanted. Samples were scanned using µCT, decalcified, incubated with phosphotungstic acid (PTA) for contrast enhancement, rescanned, and processed histologically. Results: Contrast-enhanced µCT displayed the course and branching of native bone vessels. Histologically, both central (-17%) and epiphyseal vessels (-58%) appeared smaller than in µCT scans. Hydroxyapatite cylinders were thoroughly vascularised but did not display bone formation. Grafts with a central artery had more (+58%) and smaller (-52%) vessel branches compared to grafts with a vein. Conclusions: We present a relatively inexpensive and easy-to-perform two-step method to analyse bone and vessels by µCT, suitable to assess a variety of bone-regenerative strategies.

Engineered, axially-vascularized osteogenic grafts from human adipose-derived cells to treat avascular necrosis of bone in a rat model.

BACKGROUND: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN. METHODS: SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8weeks in vivo, bone formation and vascularization were analyzed. RESULTS: Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8weeks but not in unseeded controls. Human ALU+cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks. CONCLUSION: SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells. STATEMENT OF SIGNIFICANCE: Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone.

Engraftment of Prevascularized, Tissue Engineered Constructs in a Novel Rabbit Segmental Bone Defect Model.

The gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous "incubation" (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.