Bone matrix microdamage and vascular changes characterize bone marrow lesions in the subchondral bone of knee osteoarthritis.

INTRODUCTION: Bone marrow lesions (BMLs) in the subchondral bone in osteoarthritis (OA) are suggested to be multifactorial, although the pathogenic mechanisms are unknown. Bone metabolism and cardiovascular risk factors associate with BML in epidemiologic studies. However, there are no studies at the tissue level investigating the relationship between these processes and BML. The aim of this study was to investigate the relationship between BMLs in the tibial plateau (TP) of knee OA and bone matrix microdamage, osteocyte density and vascular changes. METHODS: TP were obtained from 73 patients at total knee replacement surgery and BMLs were identified ex vivo in TP tissue using MRI. Comparator 'No BML' tissue was from matched anatomical sites to the BMLs. Quantitative assessment was made of subchondral bone microdamage, bone resorption indices, osteocyte cellularity, and vascular features. RESULTS: Several key parameters were different between BML and No BML tissue. These included increased microcrack burden (p = .01, p = .0001), which associated positively with bone resorption and negatively with cartilage volume, and greater osteocyte numerical density (p = .02, p = .01), in the subchondral bone plate and subchondral trabeculae, respectively. The marrow tissue within BML zones contained increased arteriolar density (p = .04, p = .0006), and altered vascular characteristics, in particular increased wall thickness (p = .007) and wall:lumen ratio (wall thickness over internal lumen area) (p = .001), compared with No BML bone. CONCLUSIONS: Increased bone matrix microdamage and altered vasculature in the subchondral bone of BMLs is consistent with overloading and vascular contributions to the formation of these lesions. Given the important role of BMLs in knee OA, these contributing factors offer potential targets for the treatment and prevention of knee OA.

Extracellular Vesicle-functionalized Decalcified Bone Matrix Scaffolds with Enhanced Pro-angiogenic and Pro-bone Regeneration Activities.

Vascularization is crucial for bone regeneration after the transplantation of tissue-engineered bone grafts in the clinical setting. Growing evidence suggests that mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are potently pro-angiogenic both in vitro and in vivo. In the current study, we fabricated a novel EV-functionalized scaffold with enhanced pro-angiogenic and pro-bone regeneration activities by coating decalcified bone matrix (DBM) with MSC-derived EVs. EVs were harvested from rat bone marrow-derived MSCs and the pro-angiogenic potential of EVs was investigated in vitro. DBM scaffolds were then coated with EVs, and the modification was verified by scanning electron microscopy and confocal microscopy. Next, the pro-angiogenic and pro-bone regeneration activities of EV-modified scaffolds were evaluated in a subcutaneous bone formation model in nude mice. Micro-computed tomography scanning analysis showed that EV-modified scaffolds with seeded cells enhanced bone formation. Enhanced bone formation was confirmed by histological analysis. Immunohistochemical staining for CD31 proved that EV-modified scaffolds promoted vascularization in the grafts, thereby enhancing bone regeneration. This novel scaffold modification method provides a promising way to promote vascularization, which is essential for bone tissue engineering.

Topical simvastatin improves the pro-angiogenic and pro-osteogenic properties of bioglass putty in the rat calvaria critical-size model.

Objective was to describe the effect of bioactive glass putty with and without topical simvastatin on new bone formation in critical-sized defects of rat calvaria. A calvarial bone defect was created in 20 male Wistar rats and filled with bioactive glass alone (n = 10) or combined with simvastatin (n = 10). After 4 weeks, the defects were histomorphometrically evaluated for volume fraction (Vv) of woven bone, vessel density, bioglass quantity, and inflammation. Compared to the bioglass-only group, rats treated with simvastatin had greater Vv of blood vessels (3.3% ± 0.7 vs 1.6% ± 0.1, P = .0002) and new bone (2.3% ± 0.2 vs 1.8% ± 2.5, P = .003). The Vv of the bioglass remnants in the bioglass-only group was higher than in the group treated with simvastatin (2.4% ± 0.08 vs 1.7% ± 0.3, P < .0004). Chronic inflammation was noted in 1 rat from each group. Topical simvastatin seems to improve the pro-angiogenic and pro-osteogenic properties of bioglass putty in rat calvaria critical-size defects without significant inflammation.

Platelet-rich plasma promotes angiogenesis of prefabricated vascularized bone graft.

PURPOSE: The objective was to assess the impact of platelet-rich plasma (PRP) on angiogenesis and bone formation of tissue-engineered bone in the prefabricated stage. MATERIALS AND METHODS: Both thighs of New Zealand white rabbits were used as prefabricated vascularized bone grafts using a combination of bone mesenchymal stem cells and vascular bundles in a titanium cage filled with ß-tricalcium phosphate ceramic. PRP was applied in the test group, and the same procedure was performed in the control group without the application of PRP. After 4, 8, and 12 weeks, delayed static bone scanning with technetium-99m methylene diphosphonate was performed before sacrifice, and the tissue-engineered bone samples were collected for immunohistochemical analysis using a monoclonal antibody against CD31 and histologic analysis. RESULTS: The results showed superior angiogenesis in the PRP group compared with the control group at each time point as determined by bone scintigraphy and immunohistochemical examinations. The results of histologic analysis also showed that there was more bone formation in the PRP group than in the control group at each time point. CONCLUSIONS: The application of autologous PRP was an effective strategy for increasing angiogenesis and bone formation in tissue-engineered bone and had potential significance for clinical applications.

Heparin-chitosan-coated acellular bone matrix enhances perfusion of blood and vascularization in bone tissue engineering scaffolds.

Currently, the main hurdle in the tissue engineering field is how to provide sufficient blood supply to grafted tissue substitutes in the early post-transplanted period. For three-dimensional, cell-dense, thick tissues to survive after transplantation, treatments are required for hypoxia, nutrient insufficiency, and the accumulation of waste products. In this study, a biomacromolecular layer-by-layer coating process of chitosan/heparin onto a decellularized extracellular bone matrix was designed to accelerate the blood perfusion and re-endothelialization process. The results of in vitro measurements of the activated partial thromboplastin time supported the theory that the combination of chitosan and heparin could bring both anticoagulation and hemocompatibility to the scaffold. A rabbit bone defect model was established for further evaluation of the application of this kind of surface-modified scaffold in vivo. The final results of computed tomography (CT) perfusion imaging and histological examination proved that this facile coating approach could significantly promote blood perfusion and re-endothelialization in the early post-transplanted period compared with an acellular bone matrix due to its much-improved anticoagulation property.

De novo generation of an axially vascularized processed bovine cancellous-bone substitute in the sheep arteriovenous-loop model.

BACKGROUND/AIMS: The aim of this study was to generate an axially vascularized bone substitute. The arteriovenous (AV)-loop approach in a large-animal model was applied in order to induce axial vascularization in a clinically approved processed bovine cancellous bone (PBCB) matrix of significant volume with primary mechanical stability and to assess the course of increasing axial vascularization. METHODS: PBCB constructs were implanted into 13 merino sheep together with a microsurgically created AV loop in an isolation chamber. The vascularization process was monitored by sequential magnetic resonance imaging (MRI) scans. Explants were subjected to micro-computed tomography (micro-CT) analysis, histomorphometry and immunohistochemistry for CD31 and CD45. RESULTS: Increasing axial vascularization in PBCB constructs was quantified by histomorphometry and visualized by micro-CT scans. Intravital sequential MRI scans demonstrated a significant progressive increase in perfused volume within the matrices. Immunohistochemistry confirmed endothelial lining of newly formed vessels. CONCLUSION: This study demonstrates successful axial vascularization of a clinically approved, mechanically stable bone substitute with a significant volume by a microsurgical AV loop in a large-animal model. Thus microsurgical transplantation of a tissue-engineered, axially vascularized and mechanically stable bone substitute with clinically relevant dimensions may become clinically feasible in the future.

Effects of alendronate on bone healing after tooth extraction in rats.

OBJECTIVES: Tooth extraction has been identified as an important risk factor for bisphosphonate-induced osteonecrosis of the jaw. Therefore, the main goal of this study was to determine the effects of alendronate on healing of the extraction socket and on interdental alveolar bone after tooth extraction in rats. MATERIALS AND METHODS: Animals were injected subcutaneously with vehicle or alendronate for 3-4 weeks before the first mandibular molar was extracted and these treatments were continued during post-extraction periods of 10, 21, 35 and 70 days. Mandibles were processed to evaluate healing of the extraction socket and adjacent alveolar bone by assessing bone formation, bone resorption and vascularity by histomorphometric techniques. RESULTS: Alendronate decreased new woven bone formation, blood vessel area, perimeter and number in the extraction socket at 10 days postextraction, but not at later time points. Furthermore, alendronate-treated rats had increased interdental alveolar bone volume and height only at 10 days postextraction. In addition, a 2.5-fold increase in the percentage of empty osteocyte lacunae was found in alveolar bone of alendronate-treated rats only at 10 days postextraction. CONCLUSIONS: Alendronate transiently decreases bone formation and vascularity in the extraction socket and delays the removal of interdental alveolar bone after tooth extraction in rats.

Increased nitric oxide-mediated vasodilation of bone resistance arteries is associated with increased trabecular bone volume after endurance training in rats.

Old age-associated osteoporosis is related to diminished bone blood flow and impaired nitric oxide (NO)-mediated vasodilation of the bone vasculature. Endurance exercise training restores the age-associated reduction of vasodilation in numerous vascular beds, as well as improving bone properties. The purpose of this study was to determine whether functional improvements in the bone vasculature are associated with increased bone properties after an endurance training intervention. Young adult (4-6 months) and old (24-26 months) male Fischer-344 rats remained sedentary or were trained (15 m/min walking, 15 degrees incline, 5 days/week, 10-12 weeks). Endothelium-dependent vasodilation of the femoral principal nutrient artery (PNA) was assessed in vitro using acetylcholine (ACh) and inhibitors of NO synthase (NOS) and cyclooxygenase (COX). PNA endothelium-dependent vasodilation was greater after training by 16% in young and by 24% in old animals. The NOS-mediated contribution to endothelium-dependent vasodilation was enhanced by 77% after training in old rats. Distal femur trabecular bone volume (BV/TV, %) was lower with old age in sedentary animals (young: 27+/-2%, old: 23+/-1%; P<0.05). Exercise-induced elevations in bone and marrow blood flow and the NOS signaling pathway were associated with greater BV/TV (young trained: 34+/-2%, old trained: 26+/-1%; P<0.05) relative to sedentary groups. These data demonstrate that training-induced increases in bone properties are associated with enhanced endothelium-dependent vasodilation through a NOS signaling pathway in the bone vasculature.

Bone apposition around two different sandblasted and acid-etched titanium implant surfaces: a histomorphometric study in canine mandibles.

PURPOSE: The aim of this study was to evaluate bone apposition to a modified sandblasted and acid-etched (SLA) implant surface (modSLA) in the canine mandible as compared with the standard SLA surface. MATERIAL AND METHODS: In this experimental study, all mandibular premolars and first molars were extracted bilaterally in five foxhounds. After a healing period of 6 months, each side of the mandible received six randomly assigned dental implants alternating between the standard SLA and modSLA surface. The dogs were sacrificed at 2 weeks (n=2) or 4 weeks (n=3) after implant placement. Histologic and histomorphometric analyses were then performed for each implant. RESULTS: The microscopic healing patterns at weeks 2 and 4 for the two implant types with the standard SLA and modSLA surfaces showed similar qualitative findings. New bone tissue had already established direct contact with implant surfaces after 2 weeks of healing. The mean percentage of newly formed bone in contact with the implant (BIC) was significantly greater for modSLA (28.2+/-7.9%) than for SLA (22.2+/-7.3%) (P<0.05). This difference was no longer evident after 4 weeks. An increase in BIC for both implant surface types occurred from weeks 2 to 4. This increase was statistically significant when compared with SLA at 2 weeks (P<0.05), but not when compared with modSLA at 2 weeks. CONCLUSION: The data from the present study demonstrate significantly more bone apposition for the modSLA surface than the standard SLA surface after 2 weeks of healing. This increased bone apposition may allow a further reduction of the healing period following implant placement for patients undergoing early loading procedures.

Intrinsic axial vascularization of an osteoconductive bone matrix by means of an arteriovenous vascular bundle.

BACKGROUND: The purpose of this study was to generate an autonomously vascularized hard-tissue construct suitable for microsurgical transfer. The effector of vascularization was an arteriovenous bundle inserted into a specially designed channel in the matrix. The authors also evaluated corrosion cast and intravital magnetic resonance angiography as methods for monitoring and quantifying the angiogenic response. METHODS: Thirty inbred male Lewis rats were divided into two groups. In both groups (n = 15), a disk of processed bovine cancellous bone matrix was placed into an isolation chamber. In group A, a ligated arteriovenous bundle was inserted into the biogenic matrix as a vascular carrier. In group B, there was no vascular carrier. At 2, 4, and 8 weeks after implantation, four constructs per group were evaluated by means of histology and histomorphometry and one by scanning electron microscopy of vascular corrosion casts. Micro-magnetic resonance angiography was used for intravital evaluation of the vascularized matrices. RESULTS: Vascular density was higher in group A. The capillary network in group A displayed a higher degree of maturation, with organization into vessels of different orders. Both the sprouting and intussusceptive modes of angiogenesis could be documented. Micro-magnetic resonance angiography showed a patency rate of approximately 75 percent in the bundle. CONCLUSIONS: The authors zeroed in on the issue of vascularization. The results might provide a basis for further investigations on induction of bone formation in axially prevascularized matrices. Axially vascularized bone substitutes might solve issues of availability in mass and form and provide perfusion autonomy in sites of impaired circulation.

Effect of fatigue loading and associated matrix microdamage on bone blood flow and interstitial fluid flow.

Functional adaptation of bone to cyclic fatigue involves a complex physiological response that is targeted to sites of microdamage. The mechanisms that regulate this process are not understood, although lacunocanalicular interstitial fluid flow is likely important. We investigated the effect of a single period of cyclic fatigue on bone blood flow and interstitial fluid flow. The ulnae of 69 rats were subjected to cyclic fatigue unilaterally using an initial peak strain of -6000 muepsilon until 40% loss of stiffness developed. Groups of rats (n=23 per group) were euthanized immediately after loading, at 5 days, and at 14 days. The contralateral ulna served as a treatment control, and a baseline control group (n=23) that was not loaded was also included. After euthanasia, localization of intravascular gold microspheres within the ulna (n=7 rats/group) and tissue distribution of procion red tracer were quantified (n=8 rats/group). Microcracking, modeling, and remodeling (Cr.S.Dn, microm/mm(2), Ne.Wo.B.T.Ar, mm(2), and Rs.N/T.Ar, #/mm(2) respectively) were also quantified histologically (n=8 rats/group). Cyclic fatigue loading induced hyperemia of the loaded ulna, which peaked at 5 days after loading. There was an associated overall decrease in procion tracer uptake in both the loaded and contralateral control ulnae. Tracer uptake was also decreased in the periosteal region, when compared with the endosteal region of the cortex. Pooling of tracer was seen in microdamaged bone typically adjacent to an intracortical stress fracture at all time points after fatigue loading; in adjacent bone tracer uptake was decreased. New bone formation was similar at 5 days and at 14 days, whereas formation of resorption spaces was increased at 14 days. These data suggest that a short period of cyclic fatigue induces bone hyperemia and associated decreased lacunocanalicular interstitial fluid flow, which persists over the time period in which osteoclasts are recruited to sites of microdamage for targeted remodeling. Matrix damage and development of stress fracture also interfere with normal centrifugal fluid flow through the cortex. Changes in interstitial fluid flow in the contralateral ulna suggest that functional adaptation to unilateral fatigue loading may include a more generalized neurovascular response.

Prefabrication of vascularized bone flap by demineralized bone matrix.

Demineralized bone matrix (DBM) has been reported to have osteoconductive and osteoinductive properties and has been clinically used as a bone graft alternative. In the present study we attempted to generate a vascularized bone flap by subcutaneous implantation of DBM with a vascular loop to provide blood supply in a rat model. Thirty male Sprague-Dawley rats were divided into two groups according to the presence or absence of blood supply. In the experimental group, the bone flap was created by application of 0.4 mL of DBM onto two pieces of gelatin sponge sheets between which a vascular loop was sandwiched. A prefabricated flap without a vascular loop served as the control. The flaps were biopsied at three different time intervals postoperatively (2, 4, and 6 weeks). The results showed that DBM induced subcutaneous bone formation in both of the groups. However, in the nonvascularized group, the amount of bony tissue had decreased at four postoperative weeks and continued to do so afterwards. In contrast, bone formation was active at four weeks in the vascularized group. Our study indicated that implantation of DBM can prefabricate a bone flap. Blood supply to the flap is considered a key factor of the success of this prefabrication.

[Prevascularisation strategies in tissue engineering]. / Prävaskularisationsstrategien im Tissue Engineering.

INTRODUCTION: Experiments on animals have underlined the importance of vascularisation for biointegration and functionality of any given tissue engineering device. The aim of this investigation was to dissect the angiogenetic process in the frame of axial neovascularisation of a xenogenic solid matrix. The ultimate goal of this series of studies is the application of cells onto a prevascularised matrix, with the ambition to enhance cell survival after transplantation in vivo. MATERIALS AND METHODS: We performed a study in the rat with different vascular configurations in an isolation chamber. A disc-formed biogenic hard matrix (9 x 5 mm) was encased into an isolation chamber made of Teflon. In group 1, an arteriovenous fistula (AV loop) between the femoral vessels was microsurgically constructed and was placed around the matrix (n = 15). In group 2, the vascular carrier had the form of an arteriovenous ligated pedicle (n = 15). Evaluation intervals were two, four and eight weeks after implantation. The modes of evaluation included histology, scanning electron microscopy of corrosion casts as well as intravital micro-magnetic resonance imaging (MRI). RESULTS: The arteriovenous loop as vascular carrier revealed a higher capacity for angiogenesis over the bundle configuration. The neo-fibrovascular tissue displayed minimal inflammatory elements but dense vascularisation. Scanning electron microscopy demonstrated a vivid angiogenesis with rapid evolution of the vascular bead into mature, hierarchically organised network. Micro-MRI could be used for serial investigation in terms of flow measurements and detection of thrombosis. DISCUSSION: The presence of a vascular bed prior to cell transplantation might protect against hypoxia-induced cellular death, especially at central portions of the matrix, and therefore ensure physiological function of the device. The generation of vascularised bioartificial tissue substitutes might offer new modalities of surgical reconstruction for use in reparative medicine.

Early cellular responses in cortical bone healing around unloaded titanium implants: an animal study.

BACKGROUND: A clear understanding of the early cellular events leading to osseointegration of implants is currently lacking. To gain better insight, titanium implants were inserted in a rabbit model and histologic and histomorphometric analyses were performed at early time points after insertion. METHODS: Thirty-six cylindrical implants were inserted in the tibial diaphysis of six rabbits and left to heal for 1 to 42 days. Samples were processed into paraffin or methylmethacrylate sections, on which the surface of new bone, region of altered nuclear morphology, relative surface of basic multicellular units (BMUs) and blood vessels, and bone-to-implant contact were measured. RESULTS: After coagulum formation, osteoclasts and osteoblasts were observed at the bone surface 1 week after healing. In the preexisting bone, osteocytic lacunae appeared to be devoid of cells. This region of altered nuclear morphology continued to extend for 28 days (P <0.05) after implant insertion. This expansion was accompanied by an invasion of the damaged bone by BMUs that initiated intensive bone remodeling, which reached its maximum after 4 weeks (P <0.05) but was ongoing after 6 weeks of implant insertion. CONCLUSIONS: This study evaluated the early cellular events in cortical bone surrounding titanium implants. The insertion of an implant into bone initiates a series of biologic processes, including the formation of a hematoma, altered nuclear morphology of the osteocytes surrounding the implantation site, intensive bone remodeling, and the formation of new bone, eventually leading to the osseointegration of the implant.

Prefabrication of vascularized bone grafts using recombinant human osteogenic protein-1--part 3: dosage of rhOP-1, the use of external and internal scaffolds.

In a previous study vascularized bone grafts were prefabricated with recombinant human osteogenic protein-1 (rhOP-1) using blocks of xenogenic bone mineral (BioOss) as scaffolds. The present study addressed the dosage of rhOP-1 and the combination of an external (mould) and internal scaffold (granular BioOss). In five Göttingen minipigs six prefabrication sites in the latissimus dorsi muscles were randomly assigned to groups a-f. Moulds were prepared by shaping collagen/polylactide membranes in a cylindrical form which was filled with 1g BioOss granules and rhOP-1 (a: 0; b: 50; c, f, e: 250; d: 1000 microg of rhOP-1, a-e: cylinder open to muscle, e cylinder perforated, f: cylinder open to subcutaneous fat). After 6 weeks a dose dependency of bone density (a-d: 0%; 9.4%; 15.8%; 31.1%) and vessel density (a-d: 0.3; 2.4; 7.9; 25.4 counts/view) was observed histomorphometrically. Muscular surrounding was advantageous to subcutaneous tissue. Perforations of the membranes increased vessel density and did not impair bone formation. Bone density decreased in the proximity of the polylactide membranes. The membrane material was too soft and partly collapsed and therefore needs not to be reconsidered. The use of BioOss granules with 1000 microg rhOP-1 per gram proved to be a suitable concept for prefabrication of bone transplants.

Delineating bone's interstitial fluid pathway in vivo.

Although interstitial fluid flow has been suggested to play a role in bone adaptation and metabolism, the constituents and ultrastructure of this interstitial fluid pathway are not well understood. Bone's lacunar-canalicular porosity is generally believed to be a continuous interstitial fluid pathway through which osteocytes sense external mechanical loading as well as obtain nutrients and dispose of wastes. Recent electron microscopy studies have suggested that a fiber matrix surrounds the osteocytic cell processes and fills this pericellular fluid space. However, studies injecting tracer molecules into the bone vasculature have provided conflicting results about the pore size or the fiber spacing of the interstitial fluid pathway. In addition, whether the smaller collagen-apatite porosity in adult bone is also a continuous fluid pathway is still unclear. To delineate bone's interstitial fluid pathway, four tracers of various size were injected into rats: reactive red (approximately 1 nm), microperoxidase (MP, approximately 2 nm), horseradish peroxidase (HRP, approximately 6 nm), and ferritin (approximately 10 nm). Five minutes after injection, the tibiae were harvested and processed using histological protocols optimized to minimize processing time to reduce possible redistribution of tracer molecules. The number of blood vessels and osteocytic lacunae labeled with the tracers per unit bone area was then measured for mid-diaphysial cross-sections of the tibia. While none of the tracers was detected within the mineralized bone matrix (the collagen-apatite porosity) using light microscopy, all the tracers except ferritin were found to pass through the canaliculi and appear in the osteocytic lacunae. These results indicate that while small tracers (<6 nm) readily pass through the lacunar-canalicular porosity in the absence of mechanical loading, there appears to be an upper limit or cutoff size between 6 and 10 nm for molecular movement from bone capillaries to osteocytic lacunae in rat long bone. This range of pore size contains the most likely fiber spacing (approximately 7 nm) that has been proposed for the lacunar-canalicular annular space based on the presence of a proteoglycan fiber matrix surrounding the osteocyte.

Estimation of the poroelastic parameters of cortical bone.

Cortical bone has two systems of interconnected channels. The largest of these is the vascular porosity consisting of Haversian and Volkmann's canals, with a diameter of about 50 microm, which contains a.o. blood vessels and nerves. The smaller is the system consisting of the canaliculi and lacunae: the canaliculi are at the submicron level and house the protrusions of the osteocytes. When bone is differentially loaded, fluids within the solid matrix sustain a pressure gradient that drives a flow. It is generally assumed that the flow of extracellular fluid around osteocytes plays an important role not only in the nutrition of these cells, but also in the bone's mechanosensory system. The interaction between the deformation of the bone matrix and the flow of fluid can be modelled using Biot's theory of poroelasticity. However, due to the inhomogeneity of the bone matrix and the scale of the porosities, it is not possible to experimentally determine all the parameters that are needed for numerical implementation. The purpose of this paper is to derive these parameters using composite modelling and experimental data from literature. A full set of constants is estimated for a linear isotropic description of cortical bone as a two-level porous medium. Bone, however, has a wide variety of mechanical and structural properties; with the theoretical relationships described in this note, poroelastic parameters can be derived for other bone types using their specific experimental data sets.

Mandibular reconstruction with a prefabricated vascularized bone graft using recombinant human osteogenic protein-1: an experimental study in miniature pigs. Part I: Prefabrication.

Osteogenic protein-1 (rhOP-1), also called bone morphogenetic protein-7 (BMP-7), is osteoinductive. It may be possible to reconstruct a mandible by local application of rhOP-1. However, in tumour patients this can be impossible due to scars or preceding radiotherapy. Usually vascularized bone grafts are indicated. The aim of this study was to prefabricate a vascularized bone graft in the latissimus dorsi muscle for microsurgical transplantation. In nine minipigs 600 microgram rhOP-1 were used with 8 ml xenogenic bone mineral (BioOss, Geistlich, Waldenburg, Germany) as a carrier and inserted into a pouch prepared in the M. latissimus dorsi. After 6, 12, and 24 weeks the grafts were harvested. The results were evaluated using computed tomography, histology, macro- and microangiography. A high yield of newly formed bone was obtained on the osteoconductive scaffold of the xenogenic bone. It was possible to create a vascularized osseous graft in the given shape of the BioOss blocks. In cross-sections, 68% of the scaffold was coated with new bone. The amount of new bone did not differ between the prefabrication times. Bone overgrowth was 2.1% of the graft volume. In conclusion, this study has shown that it can be possible to prefabricate a neomandible within a muscle graft, which then could be transferred for microvascular reconstruction of the mandible. Further research is required before this technique can be refined for clinical use.

A molecular analysis of matrix remodeling and angiogenesis during long bone development.

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.