Allograft Integration Research Articles

Benefits of platelet rich plasma (PRP) treatment on skin autografts and allografts in a burned patient

Wound healing is promoted by a series of growth factors, secreted from the alpha granules of the platelets. Platelet rich plasma (PRP) emerged as a clinical strategy to increase the physiologic concentration of platelets and its clinical use for a large spectrum of disorders is reported, with good results. Extensive burns represent a promising field for PRP use, those patients confronting challenging wound healing and poor long-term results. We present our initial clinical experience represented by a case of a 51-year-old male patient, severely burned, who benefited from sequential surgical treatment of deep burn wound excisions and coverage using autologous and allogenic skin grafts. PRP was added to this patient’s therapy in two stages, being injected and externally applied on the autografts in functional areas of the hands and the right upper arm region where both autografts and allografts were placed, with the exciting result of promoting allograft integration, long-term persistence and healing. Several literature data validate successful clinical applications of PRP in various therapies, including treatment of soft tissue injuries and chronic wounds, orthopedic surgery, burns, maxillo-facial surgery and elaboration of tissue-engineered products. Based on our preliminary experience and favourable reports in the literature, we encourage wider use of platelet rich plasma for immunomodulation and tissue-engineered constructs, due to its effects on local inflammatory response and role in enhancing the integration of various types of tissue grafts.

Achilles Tendon-Bone Allograft is Advantageous for Chronic Patellar Tendon Ruptures Using a Modified Fixation Technique

Objectives: A patellar tendon rupture is an extremely disabling extensor mechanism injury. This follow-up study describes the clinical and radiologic outcomes of a new modified technique for reconstructing chronic patellar tendon ruptures. Materials and Methods: From 2014 to 2018, we reconstructed 15 chronic patellar tendon ruptures using Achilles tendon-bone allograft. Clinical and radiologic outcomes were evaluated preoperatively and during the final follow-up, 2 to 7 years after reconstruction. Assessments involved the International Knee Documentation Committee (IKDC), Lysholm, and Kujala scores, as well as quadriceps muscle strength and extension lag. Radiologic results were evaluated using the Insall-Salvati index on plain radiography and allograft integration through magnetic resonance imaging. Results: At the final follow-up, the Lysholm, IKDC, and Kujala scores improved by 57, 58, and 54 points, respectively (P<0.001). The average quadriceps muscle strength improved from 2.6 (2 to 3) before the operation to 4.6 (4 to 5) at the final follow-up (P<0.001). The extension lag also improved significantly from 31.7 degrees (25 to 40) to 0.6 degrees (0 to 10). The preoperative Insall-Salvati index was 1.6 (1.5 to 1.9), which improved significantly to 1.2 (1 to 1.3) postoperatively (P<0.001). Two years after the operation, magnetic resonance imaging showed good allograft integration in all pateints. Conclusion: Combining an Achilles tendon-bone allograft with a robust fixation method led to favorable clinical and radiologic outcomes. Level of Evidence: Level IV.

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling.

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Point-of-Care Ultrasound as a Tool to Assess Wound Size and Tissue Regeneration after Skin Grafting

Chronic wounds can be difficult to heal and are often accompanied by pain and discomfort. Multiple skin substitutes or cellularized/tissue-based skin products have been used in an attempt to facilitate closure of complex wounds. Allografts from cadaveric sources have been a viable option in achieving such closure. However, early assessment of graft incorporation has been difficult clinically, often with delayed evidence of failure. Visual cues to assess graft integrity have been limited and remain largely superficial at the skin surface. Furthermore, currently used optical imaging techniques can penetrate only a few millimeters deep into tissue. Ultrasound (US) imaging offers a potential solution to address this limitation. This work evaluates the use of US to monitor wound healing and allograft integration. We used a commercially available dual-mode (US and photoacoustic) scanner operating only in US mode. We compared the reported wound size from the clinic with the size measured using US in 45 patients. Two patients from this cohort received an allogenic skin graft and underwent multiple US scans over a 110-d period. All data were processed by two independent analysts; one of them was blinded to the study. We measured change in US intensity and wound contraction as a function of time. Our results revealed a strong correlation (R2 = 0.81, p < 0.0001) between clinically and US-measured wound sizes. Wound contraction >91% was seen in both patients after skin grafting. An inverse relationship between wound size and US intensity (R2 = 0.77, p < 0 .0001) indicated that the echogenicity of the wound bed increases as healthy cells infiltrate the allograft matrix, regenerating and leading to healthy tissue and re-epithelization. This work indicates that US can be used to measure wound size and visualize tissue regeneration during the healing process.

Spondylodiscitis treated with freeze-dried bone allograft alone or combined with autograft: A randomized and blinded trial.

To compare the bone fusion of freeze-dried allograft alone versus freeze-dried allograft combined autograft in spinal instrumentation due to spondylodiscitis. A randomized prospective trial of patients with spondylodiscitis treated with surgical debridement and spinal fixation with freeze-dried bone allograft and autograft (Group 1) or freeze-dried bone allograft alone (Group 2) was performed. Patient follow-up was assessed with a CT-scan for bone fusion; consecutive serum inflammatory marker detection (C-reactive protein, [CRP], and erythrocyte sedimentation rate, [ESR]) and clinical assessment (pain, functional disability, and spinal cord injury recovery) were other outcome parameters. The primary outcome was the grade of bone allograft integration with the scale of Tan (which ranges from 1 to 4, with lower scores indicating a better fusion rate) at 1 year after surgery. A total of 20 patients were evaluated, 13 (65%) men and 7 (35%) women with a mean age of 47.2 (±14.3) years. Homogeneous distribution of demographic data was observed. A similar satisfactory bone graft fusion grade was observed in both graft groups at 1 year after surgery (p = 1.0000). Serum inflammatory markers gradually decreased in both groups after surgical intervention (CRP, p < 0.001; ESR, p < 0.01). At one-year follow-up, gradual improvement of pain, functional disability, and neurological spinal injury recovery in both graft groups were achieved. Freeze-dried allograft alone could be a therapeutic option for spinal fixation surgery due to spondylodiscitis since it achieves a satisfactory graft fusion rate and clinical improvement. Level 1. Treatment. NCT03265561.

Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration.

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair.

The Biomechanical Properties of Meshed Versus Perforated Acellular Dermal Matrices: Analysis of Surface Area and Fluid Egress

BACKGROUND: Acellular dermal matrices (ADMs) are used for soft tissue augmentation across surgical specialties. The increased use of ADMs has been particularly apparent in the setting of implant-based breast reconstruction, where matrices are used to supplement the thickness of the mastectomy skin flaps and ensure adequate soft tissue coverage of implants. However, long-term review of these devices has indicated that there is a strong correlation between seroma formation and the use of ADM. Since allograft integration and neovascularization is dependent having on direct opposition between the ADM and a vascular bed, the presence of seromas can inhibit the area of the graft which is in contact with the native tissue. As a result, most ADM products are available in a variety of meshed or perforated forms. Because of the lack of consistency between manufacture designs, we set out to determine the fluid egress properties and the increase in surface area resulting from common cut patterns. METHODS: The fluid egress properties were analyzed for three different commonly encountered commercially available ADM cut patterns: 1 meshed design and 2 distinct perforation designs. Mesh Pattern #1 was designed with 1:1 meshed cuts each measuring 1.5 mm in length. Perforation Pattern #1 was designed with 3 mm diameter perforations at a density of 0.128 perforations per cm2. Perforated Pattern #2 was designed with 3 mm diameter perforations at a density of 0.25 perforations per cm2. The surface area of these modified ADM samples was also calculated, accounting for the mesh length or the perforation diameter and frequency. Fluid egress was calculated by passing fluid through each ADM and measuring the amount of time required for complete passage. An analysis of variance was used to determine if there was a significant difference in egress properties across the three patterns. A P value of <0.05 was used to determine statistical significance. RESULTS: Meshing in a 1:1 pattern resulted in a 97.50% increase in surface area compared to the uncut product. In comparison, only a 0.30% increase resulted from Perforation Pattern #1 and a 0.59% increase resulted from Perforation Pattern #2. There was a significant difference in egress properties across the 3 cut patterns (P = 0.000). The average egress time of Mesh Pattern #1 was 1.974 seconds. The average egress time of Perforation Pattern #2 was 6.504 seconds and of Perforation Pattern #1 was 10.369 seconds. Neither donor (P = 0.249) nor graft thickness (P = 0.914) had a significant impact on the results. CONCLUSION: To our knowledge, this study is the first to directly compare clinically applicable properties between different ADM cut patterns. By comparing a variety of common manufacturer designs, ranging from simple punch-shape perforations to a full 1:1 mesh pattern, we were able to demonstrate that meshing ADM tissue significantly improves fluid egress properties and substantially increases the surface area compared to ADM tissue perforated at levels typically available on the market. Therefore, the use of meshed ADM tissue could improve the ability of the product to incorporate with the recipient, resulting in decreased complications and improved patient outcomes.

Vascular Supply and Bone Marrow Concentrate for the Improvement of Allograft in Bone Defects: A Comparative In Vivo Study

BackgroundSurgical repair of critical-sized bone defects still remains a big challenge in orthopedic surgery. Biological enhancement, such as growth factors or cells, can stimulate a better outcome in bone regeneration driven by well-established treatments such as allogenic bone graft. However, despite the surgical options available, correct healing can be slowed down or compromised by insufficient vascular supply to the injured site. Materials and methodsIn this pilot study, critical size bone defects in rabbit radius were treated with allograft bone, in combination with vascular bundle and autologous bone marrow concentrate seeded onto a commercial collagen scaffold. Microtomographical, histological and immunohistochemical assessments were performed to evaluate allograft integration and bone regeneration. ResultsResults showed that the surgical deviation of vascular bundle in the bone graft, regardless from the addition of bone marrow concentrate, promote the onset of healing process at short experimental times (8 wk) in comparison with the other groups, enhancing graft integration. ConclusionThe surgical procedure tested stimulates bone healing at early times, preserving native bone architecture, and can be easily combined with biological adjuvant.

Effect of Autogenous Bone Marrow Aspirate Treatment on Magnetic Resonance Imaging Integration of Osteochondral Allografts in the Knee: A Matched Comparative Imaging Analysis

To accurately evaluate the effects of bone marrow aspirate (BMA) augmentation on osteochondral allograft (OCA) integration on early postoperative magnetic resonance imaging (MRI) using the comprehensive Osteochondral Allograft MRI Scoring System (OCAMRISS). This imaging study compared patients who underwent OCA transplantation with and without BMA augmentation for the treatment of focal osteochondral defects in the knee performed by a single surgeon between July 2013 and July 2017. Patients were excluded if they underwent implantation of premade plugs, had an overlapping OCA configuration ("snowman" technique), or did not undergo MRI at 6months postoperatively. Patients were matched by lesion location, lesion size, age, and body mass index, as well as whether they underwent previous surgical procedures. Data were analyzed using descriptive statistics, Spearman correlation, the independent t test, the Mann-Whitney U test, and the χ2 test. A total of 58 patients (29 per group) were included in this study, with an average age of 36.4 ± 10.1years and mean body mass index of 28.6 ± 5.1. The mean size of the analyzed OCA plugs was 3.3 ± 1cm2. At an average imaging follow-up of 5.6 ± 1months, 86.2% of the grafts had achieved osseous integration at the graft-host junction and 75.9% did not show any cystic changes in the subchondral bone. No difference in any OCAMRISS subscale was seen comparing OCAs with and without BMA augmentation (P>.05). Specifically, osseous integration and subchondral cyst formation were comparable between groups (P= .128 and P= .539, respectively). OCAs showed excellent osseous integration at the graft-host junction on 6-month postoperative MRI. The treatment of OCAs with autogenous BMA did not result in superior imaging outcomes when analyzed using the OCAMRISS. Level III, case-control study.

Allograft use in anterior cruciate ligament reconstruction surgery: a review of the current literature

Anterior cruciate ligament reconstruction (ACLR) surgery plays an important role in restoring stability and function to the knee joint following ACL rupture. Owing to an increase in activity levels and sports participation, ACLR has become one of the most commonly performed procedures world-wide. Graft choice may influence clinical outcomes, and therefore the optimal graft remains widely debated. Whilst, historically, autograft tissue has been the preferred choice, the past decade has seen a steady increase in the popularity of allografts. This demand is partly driven by improvements in graft availability, procurement processes and safety; but more importantly a desire to eliminate issues related to donor site morbidity from graft harvest. Despite this, there remains controversy surrounding the use of allograft in ACLR surgery, with much of the literature demonstrating conflicting evidence on functional and survivorship outcomes. In this article we review the current literature surrounding allograft use in ACLR, from the biology of allograft integration, through to outcomes in clinical practice.

Clinical and MRI Outcomes of Fresh Osteochondral Allograft Transplantation After Failed Cartilage Repair Surgery in the Knee.

Fresh osteochondral allograft transplantation is an appealing option to address a failed cartilage repair surgical procedure, given the ability to treat large lesions and to address the subchondral osseous changes commonly seen in the revision setting. We hypothesized that osteochondral allograft transplantation after failed cartilage repair would result in low failure rates and improved function and that improved graft incorporation on postoperative magnetic resonance imaging (MRI) would correlate with a superior clinical outcome. A retrospective review of prospectively collected data was used to identify 43 patients treated with fresh osteochondral allograft transplantation after a previous cartilage repair surgical procedure and having a minimum follow-up of 2 years. Clinical outcomes were evaluated using the Short Form-36 (SF-36) score, International Knee Documentation Committee (IKDC) Subjective Knee Score, Marx Activity Scale, Knee Outcome Survey-Activities of Daily Living (KOS-ADL) Questionnaire, Cincinnati Sports Activity Score, and Cincinnati Overall Symptom Assessment. Postoperative MRI scans were obtained at a mean time of 19.7 months and were independently reviewed by a musculoskeletal radiologist using the Osteochondral Allograft MRI Scoring System (OCAMRISS). At a mean 3.5-year follow-up after osteochondral allograft transplantation, significant improvements (p < 0.05) in SF-36 Physical Function, SF-36 Pain, KOS-ADL, IKDC Subjective Knee Score, and Cincinnati Overall Symptom Assessment were seen. Over 90% of grafts remained in situ at the time of the latest follow-up, although 17 knees (40%) underwent reoperation, the majority for arthroscopic debridement or manipulation for stiffness. Body mass index (BMI) of >30 kg/m was associated with worse clinical outcomes. The mean total OCAMRISS score demonstrated poorer allograft integration in patients with graft failure, but the total score did not meaningfully correlate with clinical outcome scores. However, better individual articular cartilage appearance and osseous integration subscores were associated with better clinical outcome scores. Significant improvements in pain and function were seen following fresh osteochondral allograft transplantation after failed cartilage repair, with an overall graft survival rate of >90%. Patients with greater bone and cartilage incorporation on MRI had superior clinical outcomes, although persistent osseous edema was frequently seen. We concluded that osteochondral allograft transplantation is an effective salvage treatment after failed cartilage repair and recommend further evaluation of techniques to optimize graft integration. Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Cells, soluble factors and matrix harmonically play the concert of allograft integration.

Implantation of allograft tissues has massively grown over the last years, especially in the fields related to sports medicine. Beside the fact that often no autograft option exists, autograft related disadvantages as donor-site morbidity and prolonged operative time are drastically reduced with allograft tissues. Despite the well documented clinical success for bone allograft procedures, advances in tissue engineering raised the interest in meniscus, osteochondral and ligament/tendon allografts. Notably, their overall success rates are constantly higher than 80%, making them a valuable treatment option in orthopaedics, especially in knee surgery. Complications reported for allografting procedures are a small risk of disease transmission, immunologic rejection, and decreased biologic incorporation together with nonunion at the graft-host juncture and, rarely, massive allograft resorption. Although allografting is a successful procedure, improved techniques and biological knowledge to limit these pitfalls and maximize graft incorporation are needed. A basic understanding of the biologic processes that affect the donor-host interactions and eventual incorporation and remodelling of various allograft tissues is a fundamental prerequisite for their successful clinical use. Further, the importance of the interaction of immunologic factors with the biologic processes involved in allograft incorporation has yet to be fully dissected. Finally, new tissue engineering techniques and use of adjunctive growth factors, cell based and focused gene therapies may improve the quality and uniformity of clinical outcomes. The aim of this review is to shed light on the biology of meniscus, osteochondral and ligament/tendon allograft incorporation and how collection and storage techniques may affect graft stability and embodiment.Level of evidence V.

Biomechanical considerations are crucial for the success of tendon and meniscus allograft integration—a systematic review

This systematic review intends to give an overview of the current knowledge on how allografts used for the reconstruction of cruciate ligaments and menisci are integrated and specifically perform regarding their biomechanical function. Two reviewers reviewed the PubMed and Central Cochrane library with focus on the biomechanical integration of tendon ligament and meniscus allografts. The literature search was conducted in accordance with the PRISMA statement for reporting systematic reviews and meta-analyses. The analysed literature on tendon allografts shows that they are more vulnerable to overstretching in the phase of degradation compared to autografts as the revascularization process starts later and takes longer. Therefore, to avoid excessive graft loads, allografts for cruciate ligament replacement should be selected that exhibit much higher failure loads than the native ligaments to counteract the detrimental effect of degradation. Further, placement techniques should be considered that result in a minimum of strain differences during knee joint motion, which is best achieved by near-isometric placement. The most important biomechanical parameters for meniscus allograft transplantation are secure fixation and proper graft sizing. Allograft attachment by bone plugs or by a bone block is superior to circumferential suturing and enables the allograft to restore the chondroprotective biomechanical function. Graft sizing is also of major relevance, because too small grafts are not able to compensate the knee joint incongruity and too large grafts may fail due to extrusion. Only adequate sizing and fixation together can lead to a biomechanically functioning allograft. The objective assessment of the biomechanical quality of allografts in a clinical setting is challenging, but would be highly desirable for monitoring the remodelling and incorporation process. Currently, indicators like ap-stability after ACL reconstruction or meniscal extrusion represent only indirect measures for biomechanical graft integration. These parameters are at best clinical indicators of allograft function, but the overall integration properties comprising e.g. fixation and graft stiffness remain unknown. Therefore, future research should e.g. focus on advanced imaging techniques or other non-invasive methods allowing for in vivo assessment of biomechanical allograft properties.

Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction.

Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between theligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.

Reconstruction With Rib Graft for Acetabular Revision in Pelvic Discontinuity

Acetabular osteolysis associated with socket loosening is one of the main long-term complications of total hip arthroplasty. In case of major bone loss, even metal ring or cage in association showed poor long-term outcome. To promote allograft bone consolidation and acetabular stability, we have associated a rib graft and column fixation to reconstruct the acetabular surface. Here, we describe 8 cases and the technical details of major acetabular reconstruction using revision cup with autologous rib graft. The main advantages of this procedure are the aid in allograft integration and partial unload of the allograft. Our preliminary results using rib bone graft indicate a good allograft consolidation and remodeling. The small number of patients does not allow conclusions about the efficacy of this technique but it offers another solution for extreme severe bone loss.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells.

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Effect of VEGF-A165 addition on the integration of a cortical allograft in a tibial segmental defect in rabbits.

Long-bone segmental defects caused by infection, fracture, or tumour are a challenge for orthopaedic surgeons. Structural allografts are sometimes used in their treatment but their poor biological characteristics are a liability. The objective of this study was to determine whether the addition of recombinant vascular endothelial growth factor-A (VEGF) to a structural allograft improved its integration into a rabbit tibial segmental defect in a non-union model. Tibial segmental defects were filled with heat sterilized allogenic tubular tibiae sections and then stabilized with a screw plate. In the VEGF treatment group (n = 6 tibiae), 2 μg of VEGF added to a 50 μl matrigel solution was inserted into the allograft cavity. In the control group (n = 6 tibiae), only matrigel was added. After 12 weeks, macroscopic and microscopic analysis, radiographs, and computerized micro-tomography (micro-CT) were performed. If allograft consolidation was present, a torsional resistance analysis was performed. Addition of VEGF to the allograft decreased the rate of osteosynthesis failure compared with the control group (1/6 vs. 5/6, p = 0.08), increased trabecular continuity evaluated by micro-CT in the bone-allograft interphases (8/12 vs. 2/12, p = 0.036) and histological trabecular continuity (7/12 vs. 0/12, p = 0.0046). Full consolidation was observed in three tibiae of the VEGF group and one in the control group (differences not significant); however, torsional resistance showed no significant differences (n.s.). Addition of VEGF to a structural allograph inserted into a rabbit tibial segmental defect increased allograft integration rate. Further research in this direction might help clinicians in dealing with large bone defects.

PTH Promotes Allograft Integration in a Calvarial Bone Defect

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

Quantifying Massive Allograft Healing of the Canine Femur In Vivo and Ex Vivo: A Pilot Study

Allograft integration in segmental osseous defects is unpredictable. Imaging techniques have not been applied to investigate angiogenesis and bone formation during allograft healing in a large-animal model. We used dynamic contrast-enhanced (DCE)-MRI and cone beam (CB)-CT to quantify vascularity and bone volume in a canine femoral allograft model and determined their relationship with biomechanical testing and histomorphometry. Femoral ostectomy was performed in three dogs and reconstructed with a 5-cm allograft and compression plate. At 0.5, 3, and 6 months, we performed DCE-MRI to quantify vascular permeability (Ktrans) and perfused fraction and CB-CT to quantify bone volume. We also performed posteuthanasia torsional testing and dynamic histomorphometry of the grafted and nonoperated femurs. DCE-MRI confirmed the avascular nature of allograft healing (perfused fraction, 2.08%-3.25%). CB-CT demonstrated new bone formation at 3 months (26.2, 3.7, and 2.2 cm(3)) at the graft-host junctions, which remodeled down at 6 months (14.0, 2.2, and 2.0 cm(3)). The increased bone volume in one subject was confirmed with elevated Ktrans (0.22) at 3 months. CB-CT-identified remodeled bone at 6 months was corroborated by histomorphometry. Allografted femurs recovered only 40% of their strength at 6 months. CB-CT and DCE-MRI can discriminate differences in angiogenesis and bone formation in the canine allograft model, which has potential to detect a small (32%) drug or device effect on biomechanical healing with only five animals per group.

Osteogenic Protein-1 Associated with Mesenchymal Stem Cells Promote Bone Allograft Integration

Limited incorporation and modest bone remodeling can cause allograft failure. We investigated whether mesenchymal stem cells (MSCs) and osteogenic protein-1 (OP-1) can improve allograft integration. A 3-cm full-size intercalary bone defect was created in the mid-diaphysis of the metatarsal bone of the sheep and it was replaced with an allograft alone (control group), or with MSCs (MSC group), OP-1 (OP-1 group), or MSCs and OP-1 (MSC + OP-1 group). Radiographic results showed a faster and complete integration of the allograft in the MSC + OP-1 group. Histology demonstrated that the amount of new bone was significantly greater inside the graft and a longer vessel penetration in the MSC + OP-1 group than in others. Mechanical strength of the allograft was not compromised by the high rate of bone remodeling. These results demonstrated that the association of MSCs and OP-1 improve bone allograft integration promoting an almost complete bone restoring.