Growth and survival of vascularized and nonvascularized membranous bone: an experimental study

Although nonvascularized membranous bone grafts to the craniofacial skeleton demonstrate improved survival over similar grafts of endochondral origin, the comparative fate of vascularized membranous grafts is unknown. It is also unknown whether onlay membranous bone grafts in immature animals have the ability to grow. To examine these questions, a model was developed in New Zealand white rabbits in which a segment of the zygomatic arch was transferred to the subjacent mandible as either a vascularized or nonvascularized transfer. At harvest 16 weeks later, residual graft volume and bone architecture were analyzed. Results demonstrate no improved survival for vascularized membranous grafts in adult animals (n = 7), while in the immature animals (n = 6), growth of the vascularized bone transfers was documented. We conclude that in the majority of instances in craniofacial reconstruction, nonvascularized onlay membranous grafts are to be preferred. Specific instances for the use of vascularized transfers will be discussed.     

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.     

Differential patterns of incorporation and remodeling among various types of bone grafts

The early (3 months) and later (6 months) patterns of incorporation and bone formation have been evaluated histomorphometrically for different types of bone grafts; that is, vascularized and nonvascularized autografts with and without ciclosporin, and vascularized and nonvascularized dog leukocyte antigen (DLA)-mismatched allografts with and without ciclosporin. The vascularized bones were superior to the nonvascularized ones in healing and remodeling their grafted segments. In the autograft bones, ciclosporin did not alter the incorporation process 3 months after transplantation but delayed and increased the remodeling activities in the long run (6 months). Nonvascularized allografts underwent vigorous resorption, and were markedly porotic. Ciclosporin administration significantly reduced resorption and enhanced remodeling in nonvascularized allografts. The remodeling of allografts was similar to that of autografts in the presence of ciclosporin, but stopped soon after the administration of ciclosporin ceased.     

Microsurgical toenail transfer to the hand

Free nonvascularized toenail grafts have been used to reconstruct congenital or traumatic nail defects of the thumb or finger. Unfortunately, these transfers often result in deformity or atrophy. To avoid these undesirable results, microsurgical free vascularized toenail transfer was performed in 10 patients, 3 for congenital nail absence and 7 for traumatic nail defects. Patient age averaged 17 years (range 2 to 32 years). In contrast with previous reports, the whole big or second toenail complex without pulp was used in reconstruction. All 10 nails were successfully transferred with complete survival. No digits required reexploration. There were no donor- or recipient-site problems. Follow-up averaged 3 years, with a range of 14 months to 5 years and 4 months. Appropriate nail growth occurred in the congenital patients. No atrophy of the nail complex was found as long as sufficient bony support was present (9 of 10 cases). Whole free vascularized toenail transfers for reconstruction of congenital and traumatic nailbed defects achieve excellent aesthetic results while maintaining normal hand function.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

Vascularized versus nonvascularized nerve grafts: an experimental structural comparison

This study compared the success of nerve regeneration through conventional nonvascularized and vascularized nerve grafts in the sciatic nerve of rats. The number or size of regenerated axons between the two grafts was not significantly different. In addition, the ratio of axonal diameter to total diameter of the nerve, a measurement linearly related to conduction velocity, was not significantly different in the two groups. Thicker myelin sheaths were found around axons in the nonvascularized nerve grafts.     

A biomechanical comparison of vascularized and conventional autogenous bone grafts

Vascularized and conventional autogenous rib grafts were used to reconstruct 6-cm ulnar defects in the forelegs of the nine dogs. Each dog served as its own control. Biomechanical torsional testing of the grafted ulnas showed that vascularized grafts were 234 percent stronger than the conventional grafts. Bone toughness (energy absorbed) was 483 percent greater in the vascularized grafts, and elastic modulus and proportional limits were 263 and 246 percent greater, respectively. We conclude that vascularized bone grafts are significantly stronger than conventional autogenous bone grafts after 3 months of healing in the dog ulna model.     

Growth potential in onlay bone grafts: a comparison of vascularized and free calvarial bone and suture bone grafts

Vascularized bone grafts are characterized by a viable cell population with osteogenic potential. These features suggest that continued growth can be anticipated following vascularized membranous bone transfer in a growing craniofacial skeleton. The present paper compares the potential for appositional bone growth in vascularized and free calvarial onlay bone grafts. In seven 8-week-old beagles, growth was assessed by direct caliper measurements of graft dimensions intraoperatively and 16 weeks postoperatively. Vascularized grafts demonstrated a 50 to 60 percent increase in size in all dimensions compared to 10 to 20 percent growth in free grafts (p less than 0.01). Microradiography revealed preservation of calvarial bony architecture and minimal resorption in vascularized grafts, while triple-fluorochrome labeling confirmed subperiosteal appositional bone formation. Free grafts were characterized by significant resorption and a delay in subperiosteal bone formation.     

Functional rehabilitation of the atrophic mandible and maxilla with fibula flaps and implant-supported prosthesis

Historically, nonvascularized bone grafts have been the standard treatment for severe mandibular and maxillary atrophy, followed by immediate or delayed implant placement. Extreme atrophy is an unfavorable biological and mechanical location for nonvascularized autologous bone transplants. The authors present the results of a multidisciplinary treatment protocol for rehabilitation of extreme mandibular and maxillary atrophy by use of the vascularized fibular flap. This protocol includes bone augmentation, implant surgery, soft-tissue management, and prosthetic restoration. Since 1993, 18 patients with a mean age of 47.5 years presented with extreme mandibular and/or maxillary atrophy and underwent alveolar crest augmentation with vascularized fibular flaps. Bone healing was achieved in 17 of the 18 patients. Seventy-three osteointegrated implants were inserted in 12 of 17 fibular flaps. Altogether, 62 implants were loaded and 11 dental prostheses were made. Average follow-up of the loaded implants was 41 months. The success rate of loaded implants was 100 percent. The authors strongly recommend the use of the fibular bone flap when dealing with extreme atrophy of the mandible and maxilla and suggest the protocol outlined in this review.     

Free nonvascularized composite nail grafts: an institutional experience

Free, nonvascularized composite nail grafts have been reported as a successful method to reconstruct nail deformities due to congenital anomalies or traumatic defects. The authors performed a decade review of their experience with nine patients who had had 10 free, nonvascularized composite nail grafts. Patient demographics, mechanism of injury, timing, site of reconstruction, and amount of nail to be replaced were all recorded. Results of nail growth in reconstructed nails were judged as excellent, good, fair, or poor on the basis of the appearance of the nail. The majority of reconstructed nails had half or more of the nail bed replaced. The 10 cases (mean follow-up of 1.8 years) that were reported had two excellent, three good, two fair, and three poor outcomes. There was no apparent relation between the successful outcome of the procedure and patient age, timing of reconstruction, or amount of nail bed replaced. Although the authors' experience suggests the unpredictable nature of this type of graft, it should be considered for patients who desire nail reconstruction and are not candidates for ablative or vascularized nail complex transfer procedures.     

The effects of vascularity and cyclosporin A on the mechanical properties of canine fibular autografts.

We studied the biomechanical behavior of orthotopic canine autografts as influenced by vascularized supply and the administration of cyclosporin A at three months and six months post-surgery. The model was the proximal 8 cm of the fibula in young adult dogs. In vascularized grafts, blood supply was re-established by microvascular re-anastomosis. Experimental controls were sham-operated and unoperated bones. Mid-graft test sections were subjected to loading-to-failure in torsion to determine the strength and stiffness. In both three- and six-month groups, vascularized grafts were significantly stronger and stiffer than contralateral nonvascularized grafts. Vascularized grafts were not significantly different from sham-operated bones. A 30-day regimen of cyclosporin A was found to have no measurable effect on mechanical properties for any individual treatment group. The results indicate that re-established blood supply can be a major factor in maintaining the mechanical integrity in large-segment cortical autografts.     

Bone grafts: a radiologic, histologic, and biomechanical model comparing autografts, allografts, and free vascularized bone grafts

We developed an experimental model to compare the efficacy of free vascularized bone grafts, conventional segmental autografts, matchstick autografts, and fresh segmental allografts in terms of their ability to reconstruct a 7-cm segmental diaphyseal defect created in the canine femur. Forty-five adult mongrel dogs were studied and followed for 6 to 12 months prior to sacrifice. Evaluation included radiologic assessment of graft incorporation and hypertrophy, histology, and biomechanical testing. These studies indicated that microsurgically revascularized autografts were superior to all other groups in terms of early incorporation, hypertrophy, and the highest mechanical strength to failure. Union of the bone graft to the recipient femur was achieved by 6 months in 25 of 26 autografts, and no difference in union rate was seen within the autograft group. However, only two of five allografts achieved bony union during this time interval. Arteriography, microangiography, fluorochrome, and histologic studies all supported the concept that microsurgically revascularized grafts, when successful, maintain their viability. However, the premise that all osteocytes survive in a successfully revascularized bone graft is open to question. While decalcified sections showed that all microsurgically revascularized grafts maintained normal viability in the central marrow and cancellous portions compared with the other three groups, the viability of cortical bone in the vascularized autografts was less clear. Undecalcified fluorochrome sections suggested that circulation was not preserved in all portions of the cortex. Revascularization of the nonvascularized autografts was complete at 3 months, while, in the avascular allografts, the process was not complete at 6 months.     

Influences of osteoclast deficiency on craniofacial growth in osteopetrotic (op/op) mice

It is well known that the defect in bone resorption in osteopetrotic (op/op) mice brings about deformation of the cranium and failure of tooth eruption. However, the influences on longitudinal growth of the craniofacial skeleton have not been elucidated. This study was thus conducted to examine craniofacial morphology and longitudinal changes in the op/op mice by means of morphometric analysis with lateral cephalograms. Lateral cephalograms, taken every 10 days from 10- to 90-day-old mice, were analyzed on a personal computer for 11 measurement items. For the nasal bone region, the most prominent differences were found between the op/op and normal mice. The anterior cranial base and occipital bone height presented almost equivalent growth changes in both the op/op and normal mice. The size of mandible, meanwhile, was significantly smaller in the op/op mice than in the normal controls. The gonial angle was also significantly larger in the op/op mice than in the normal mice throughout the experimental period. Thus, substantial differences in craniofacial growth were demonstrated in various areas of the craniofacial complex, which are assumed essentially due to the lack of osteoclastic bone resorption during growing period. Since the difference became more prominent in the anatomic regions relevant to the masticatory functions, it would be a reasonable assumption that reduced masticatory function is also a key determinant for the less-developed craniofacial skeleton in the op/op mouse.     

The effect of total maxillary advancement on facial growth.

The effect of total maxillary advancement on the growing skull was examined in 25 pigs, subjected to facial operations of varied severity at the age of 5 weeks (elevation of periosteum, simple osteotomy, and osteotomy immobilized by wire fixation, with or without bone grafts) and in controls. When the animals were killed 8 months later, the body weight had increased tenfold and the skull size had doubled. In 5 pigs whose maxillae were advanced 10 mm by bone grafts, the relative increase in length was not maintained. The overall skull length showed no difference from the control measurements. The extent of the growth in the area adjacent to the osteotomy was less than in the controls, but the same as in pigs subjected to periosteal elevation only. Scattered areas of bone damage were seen with no consistent pattern. Incision of the periosteum over the frontal bone decreased the local blood supply, increased the adherence of the periosteum to the bone, and affected bone growth locally.     

Vascular knee allograft transplantation in a rabbit model

Using a rabbit model in which vascularized knee autograft transplantation was successful, vascularized knee allograft transplants survived an average of 9 days, as determined by serial bone scan. The longest surviving allograft was one of 3 months. Immunosuppression and irradiation did not significantly increase survival. Both vascularized and nonvascularized allografts elicited a second-set skin graft response but no histologic evidence of rejection. This suggests that joint allografts are clearly immunogenic but do not undergo the same destructive rejection process with a clear end point seen with soft-tissue grafts. Donor vessels did show a classic rejection picture with severe intimal damage presumably predisposing to vessel thrombosis and graft loss. Vascular rejection, therefore, limited joint allograft survival. Immediate vascularization of the allograft with subsequent limited survival does not enhance host revascularization and creeping substitution at 1, 3, or 6 months. These findings do not suggest clinical applicability for vascularized joint allograft transplantation at this time. Future experimental studies should employ genetically defined models.     

Revascularization of cranial versus iliac crest bone grafts in the rat

Full-thickness cranial (membranous) and split-thickness iliac crest (endochondral) onlay bone grafts were placed subperiosteally without fixation onto the snout (membranous) and tibia (endochondral) in 30 rats. The animals had been divided into three equal groups in which the bone grafts had been demineralized, autoclaved, or used fresh. Recipient sites were harvested at 7 and 14 days at the snout and 14 days at the tibia, and revascularization was studied utilizing silicone rubber injection and a gridcounting technique. Endochondral grafts were found to have quantitatively greater revascularization than membranous grafts in all three groups at both sites (p less than 0.005). There was generally no statistically significant difference in revascularization between fresh and demineralized grafts, but vessel ingrowth was significantly decreased in autoclaved implants as compared with fresh grafts. Differences in graft architecture are theorized to account for the disparity in revascularization in endochondral and membranous grafts. Angiogenic and chemotactic factors are thought to play a role in the similarities and differences in revascularization among fresh, demineralized, and autoclaved bone.     

Tumor Necrosis Factor Improves Vascularization in Osteogenic Grafts Engineered with Human Adipose-Derived Stem/Stromal Cells

The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.     

Development and Involution of Thymus Grafts in Rats with reference to Age and Sex

Two weeks after grafting, whole thymus implants had the characteristic appearance of a normal young thymus. They increased in weight until 6 weeks post-operation and then underwent involution. Maximum weights attained by grafts in male hosts were significantly higher than those attained in female hosts. Initial rates of graft involution were higher in males than in females. Weights attained by grafts in young hosts of either sex reached maxima that were significantly higher than those attained by grafts of the same age in old hosts. The number of thymuses grafted separately into a single host did not influence the weight of the host's own thymus or the mass of individual grafts. However, when multiple grafts were linked together on transplantation, they behaved as an individual graft with respect to general morphology, growth rate and maximum size. With respect to growth and involution, it is suggested that thymic lymphopoiesis is mainly regulated by intrinsic factors emanating from the tissue which survives implantation. Further, the regulators appear to be clone-specific.     

Interphalangeal joint and tendon forces: normal model and biomechanical consequences of surgical reconstruction

Soft tissue reconstructive surgery for rheumatoid-related proximal interphalangeal joint deformities frequently fails to produce the long-term predicted results. Detailed information on the biomechanics of this joint, under both normal and pathological conditions, is required to assess the efficacy of such surgical intervention. A biomechanical model of the proximal interphalangeal joint has been developed to investigate tendon and joint loading during real life three-dimensional activities. Based on a rigid body mechanics approach, the model uses high resolution MRI scans to obtain anatomical tendon and bone geometries in conjunction with three-dimensional kinematic and loading data. The model incorporates an optimisation routine which minimises overall maximum tendon stress in the eight individual elements considered. Radial and ulnar joint force components are included at the proximal interphalangeal joint level. Two simulated pathological versions of the mathematical model are developed to accommodate the altered anatomic relationships after tendon reconstructive surgery. Joint forces of up to 450N and common usage of the extensor mechanism during normal pinching and grasping activities are predicted. The ulnar lateral bands of the extensor tendon are generally loaded to a greater extent than the radial bands. Extensor tendon and joint forces in the simulated pathological models are significantly higher than those in the normal model. Combined with the poor tendon quality of rheumatoid arthritis patients generally, these amplified internal forces may lead to further joint deformation.     

Radiographic analysis of growth in pediatric microsurgical toe-to-hand transfers

Microsurgical toe-to-hand transfers may provide improved hand function in children with absent digits. To date, documentation of the growth potential of these transferred digits has not been performed. This study reviewed the authors' series of pediatric toe-to-hand transfers, with specific attention paid to measuring growth by radiographic analysis. From 1995 to 2000, 23 toe-to-hand transfers were performed in 18 children. Age at time of transfer ranged from 2.8 to 13 years. Indications included constriction band syndrome, transverse deficiency, longitudinal deficiency, traumatic amputation, and vascular malformation. The transfers were successful in 22 of 23 procedures (96 percent success rate). Radiographic analysis of growth was performed using three criteria: (1) appearance of open epiphyseal plates, (2) comparison with preoperative radiographs, and (3) comparison with radiographs of the contralateral control toe. Epiphyseal plates remained open on postoperative radiographs in 27 of 28 phalanges (96 percent) at a mean of 12 months' follow-up (range, 1 to 36 months). The preoperative foot radiographs were compared with serial radiographs of the transferred toe over time. In 10 toe transfers with follow-up greater than 6 months (mean, 21 months), nine patients had increased bony length in the transferred digit. In four patients, radiographs of the toe transfer were compared with radiographs of the corresponding toe on the opposite foot. With a mean follow-up of 29 months, all patients had equal length measurements of the toe transfer with the contralateral toe control. These data provide objective evidence that digital growth potential is preserved in toe-to-hand transfers. Furthermore, this bone growth is comparable with that of the corresponding toe on the contralateral foot. Therefore, microsurgical toe-to-hand transfers may provide children with extra digits that maintain growth and improve hand function.