Prefabrication of bone by use of a vascularized periosteal flap and bone morphogenetic protein.

The purpose of this pilot study was to prefabricate a vascularized bone graft by using a vascularized periosteal flap containing osteoprogenitor cells, a structural matrix, and recombinant human bone morphogenetic protein-2 (rhBMP-2). In a rat model, a periosteal flap vascularized by the saphenous artery and vein was dissected off the medial surface of the tibia. This flap consisted of three layers-periosteum, muscle, and fascia-and was tubed on itself to form a watertight chamber that was then transferred on its vascular pedicle to the groin. A total of 78 vascularized periosteal chambers were constructed in 39 animals and divided into 10 groups. In group 1, the periosteal chamber was left empty. Groups 2, 3, and 4 consisted of the periosteal flap and rhBMP-2, but in group 3, the proximal vascular pedicle was ligated, and in group 4, the flap was harvested without the periosteal layer and turned inside out. Groups 5 through 10 consisted of the vascularized periosteal flap containing several different structural matrices (calcium alginate spheres, polylactic acid, or demineralized bone matrix) with or without rhBMP-2. Animals were killed at 2, 4, or 8 weeks in each group. The presence and density of any new bone formation was evaluated both radiologically and histologically. Significant bone formation was seen only in those periosteal flaps containing rhBMP-2 and either the calcium alginate or polylactic acid matrix. New bone formation increased both radiologically and histologically from 2 weeks to 8 weeks only in the periosteal flaps containing the polylactic acid matrix and rhBMP-2. This preliminary study therefore suggests that four factors-blood supply, osteoprogenitor cells in the periosteal layer, a biodegradable matrix, and rhBMP-2-are required for optimal prefabrication of a vascularized bone graft.     

Peripheral neovascularization of muscle and musculocutaneous flaps in the pig.

Late loss of free muscle flaps following surgical or accidental trauma to the dominant vascular pedicle has been reported. In this study, time-dependent ligation of the dominant vascular pedicle was undertaken in denervated latissimus dorsi musculocutaneous or muscle-only island flaps in the pig. Muscle flaps were covered with a skin graft, and silicon rubber sheets were inserted between the flaps and their bases to simulate a poorly vascularized bed. Hemodynamic and viability studies were then performed using intravenous fluorescein (skin viability), tetrazolium blue (muscle viability), and radiolabeled 15-micron microspheres (capillary blood flow). Blood flow did not change in acutely raised musculocutaneous flaps (n = 10) but was significantly elevated in acutely raised muscle-only flaps (n = 10), suggesting that the skin paddle may steal blood flow from the underlying muscle in musculocutaneous flaps. Peripheral neovascularization at 1 day to 8 weeks was assessed (n = 30). Viability increased during the first week of revascularization and was not different in musculocutaneous and muscle-only flaps. Revascularization of muscle-only flaps was enhanced compared with musculocutaneous flaps in the 2- to 8-week period.     

Vascularized outer-table calvarial bone flaps

Based on an anatomic study of the vascularization of the calvarium in cadavers, a technique for the transfer of vascularized outer-table calvarial bone has been developed. The outer table of the calvarium receives numerous small perforators from its overlying periosteum. The periosteum is continuous with a distinct fascial layer overlying the temporal aponeurosis which we have termed the innominate fascia. Because of a network of anastomosing vessels from proximal branches of the superficial temporal artery and perforating branches of the deep temporal artery, the outer table of the calvarium can be carried on a pedicle which contains the temporal aponeurosis, innominate fascia, and periosteum. Thirty-seven vascularized outer-table calvarial bone flaps have been performed for a variety of craniofacial reconstructive deformities. Remarkable stability and lack of resorption have led the authors to favor this method of reconstruction particularly in poorly vascularized or previously infected recipient beds.     

Prefabricated sensate myocutaneous and osteomyocutaneous free flaps: an experimental model. Preliminary report

Principles of neovascularization have been reported for the successful creation of a variety of muscle and bone free flaps. This study demonstrates a simple and effective technique for construction of prefabricated sensate myocutaneous and osteomyocutaneous free flaps in a rat model. These experiments were carried out in 20 Sprague-Dawley male rats. In half the animals, a sensate myocutaneous flap was constructed by sandwiching the superficial inferior epigastric vessels between a laterally based external abdominal oblique muscle flap and a laterally based skin flap served by an identified cutaneous nerve. A similar preparation included a piece of iliac crest bone. Two to three weeks later, now neovascularized by the sandwiched vessels, the flaps were harvested and transferred as free flaps with high reliability. An increased number of potential donor sites, the versatility of design, and the ability to customize flaps to the specific recipient-site needs are proffered.     

Anatomic basis for vascularized outer-table calvarial bone flaps

The vascularization of the scalp and calvarium was studied in cadavers to better define the design of vascularized split- or full-thickness calvarial bone flaps. Selective dye injections of the superficial temporal and internal maxillary arteries established a horizontal and vertical network of vessels within and between each layer of the scalp. The periosteum of the frontoparietal region continues over the temporal aponeurosis as a separate, distinct layer, the innominate fascia, which is irrigated by numerous proximal branches of the superficial and deep temporal arteries. The periosteum can sustain the outer table of the calvarium by means of multiple small, vertical perforators. Between the periosteum and the outer table is a thin areolar layer of subperiosteum which continues beneath the temporal muscle. We feel that vascularized outer-table calvarial flaps can safely be pedicled using only the temporal aponeurosis, innominate fascia, and periosteum without including the galea or temporal muscle.     

One-stage reconstruction of composite bone and soft-tissue defects in traumatic lower extremities

Management of bone loss that occurs after severe trauma of open lower extremity fractures continues to challenge reconstructive surgeons. Sixty-one patients who had 62 traumatic open lower extremity fractures and combined bone and composite soft-tissue defects were treated with the following protocol: extensive debridement of necrotic tissues, eradication of infection, and vascularization of osteocutaneous tissue for one-stage bone and soft-tissue coverage reconstruction. The mechanism of injury included 49 motorcycle accidents (80.3 percent), five falls (8.2 percent), three crush injuries (4.9 percent), two pedestrian-automobile accidents (3.3 percent), and two motor vehicle accidents (3.3 percent). The bone defects were located in the tibia in 49 patients (79 percent; one patient had bilateral open tibial fractures), in the femur in seven patients (11.3 percent), in the calcaneus bone in four patients (6.5 percent), and in the metatarsal bones in two patients (3.2 percent). The size of soft-tissue defects ranged from 5 x 9 cm to 30 x 17 cm. The average length of the preoperative bony defect was 11.7 cm. The average duration from injury to one-stage reconstruction was 27.1 days, and the average number of previous extensive debridement procedures was 3.4. Fifty patients had vascularized fibula osteoseptocutaneous flaps, six had vascularized iliac osteocutaneous flaps, and five patients had seven combined vascularized rib transfers with serratus anterior muscle and/or latissimus dorsi muscle transfers. One patient received a second combined rib flap because the first combined rib flap failed. The rate of complete flap survival was 88.9 percent (56 of 63 flaps). Two combined vascularized rib transfers with serratus anterior muscle and latissimus dorsi muscle flaps were lost totally (3.2 percent) because of arterial thrombosis and deep infection, respectively. Partial skin flap losses were encountered in the five fibula osteoseptocutaneous flaps (7.9 percent). Postoperative infection for this one-stage reconstruction was 7.9 percent. Excluding the failed flap and the infected/amputated limb, the primary bony union rate after successful free vascularized bone grafting was 88.5 percent (54 of 61 transfers). The average primary union time was 6.9 months. The overall union rate was 96.7 percent (59 of 61 transfers). The average time to overall union was 8.5 months after surgery. Seven transferred vascularized bones had stress fractures, for a rate of 11.5 percent. Donor-site problems were noted in six fibular flaps, in two iliac flaps, and in one rib flap. The fibular donor-site problems were foot drop in one patient, superficial peroneal nerve palsy in one patient, contracture of the flexor hallucis longus muscle in two patients, and skin necrosis after split-thickness skin grafting in two patients. The iliac flap donor-site problems were temporary flank pain in one patient and lateral thigh numbness in the other. One rib flap transfer patient had pleural fibrosis. Transfer of the appropriate combination of vascularized bone and soft-tissue flap with a one-stage procedure provides complex lower extremity defects with successful functional results that are almost equal to the previously reported microsurgical staged procedures and conventional techniques.     

De novo induction of island capsule flap by using two silastic sheets: Part 1. Generation

A new experimental model for de novo generation of an axial pattern island flap has been designed in a rat model. The purpose of this study was to make a sufficient vascular carrier, as an island capsule flap, with only vascular pedicles and addition of collagen fibers induced by foreign-body reaction. The femoral arteriovenous bundle was isolated and sandwiched between two 2.5 x 1.5 cm Silastic sheets. Eight weeks later, as a delay procedure, femoral vessels were ligated at the distal end of the Silastic sheets and the four margins of the sheets were divided except for the vascular pedicle. This capsule flap was raised as a secondary island flap connected only by its vascular pedicle, then it was sutured back in place. Ten days after the delay procedure, the upper Silastic sheet was removed and a full-thickness skin graft was performed on the capsular island flap. Animals were killed at 80 days. A total of 40 axial pattern capsulocutaneous flaps from 20 Sprague-Dawley rats were successfully achieved. Pathologic study revealed neovascularization, and abundantly impregnated vascular structures near the pedicle were observed with randomly developed collagen fibers. The skin graft took 100 percent on this newly formed capsular flap; therefore, the capsule structure was able to survive on its own and support skin grafts. This experiment, by using an isolated femoral artery and vein as the main pedicle, led to the formation of a capsule flap through a normal foreign body reaction between two Silastic sheet implants. This new flap can be used as a reliable vascular carrier for various needs with minimal donor morbidity.     

Comparison of residual osseous mass between vascularized and nonvascularized onlay bone transfers

Composite flaps containing vascularized frontal bone were transferred on muscle pedicles in immature rabbits. Vascular continuity was maintained on one side and interrupted on the other. Bone weights at 16 weeks following transfer were compared with those of unoperated controls. The conventional bone graft demonstrated significant reduction in osseous mass. The vascularized bone maintained its mass compared with unoperated controls. Vascularized bone transfer appears to be the preferred surgical technique whenever possible.     

Molded vascularized osteoneogenesis: a preliminary study in rabbits

The clinical application of free vascularized bone grafting techniques in reconstructive surgery has limitations with respect to available donor sites and configuration of the graft. In this study, we were able to produce a vascularized bone graft with a predetermined size and shape by the use of titanium chambers implanted into the groins of rabbits. Forty adult New Zealand white rabbits were included in this study. In all animals, bone fragments were harvested from the iliac crests and placed within a titanium chamber. The chamber was positioned in the groin with the saphenous vessels passing through the chamber longitudinally in contact with the bony fragments. In half the animals, a segment of periosteum was harvested from the tibia and sutured around the vessels with the cambium layer facing the bone fragments. Sacrifices were performed at 4, 8, 12, and 16 weeks to examine the chamber contents. At all time periods, the contents completely filled the chamber, and the saphenous vessels remained patent. Histologic evaluation showed remodeling of the bone fragments as early as 4 weeks concomitant with a sprouting vascular network demonstrable with microangiography. Fluorochrome bone labels documented new bone formation within the chamber. These changes were progressive to week 12, but the 16-week specimens appeared to be undergoing resorption.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

The thigh flap: an osteomyocutaneous free-flap model in the rat

A new experimental model for free-flap transfer has been developed in the rat. This "thigh flap" is an osteomyocutaneous free flap of bone (femur), muscle (thigh), and skin (groin) based on the femoral vessels. The flap is harvested from the left groin and thigh of an inbred female rat and is transferred to a subcutaneous pocket in the left groin of a male rat of the same inbred strain. The femoral vessels supplying the flap are anastomosed end-to-end with the femoral vessels of the recipient. Thirty flaps have been transferred, with 5 technical failures. Three of the remaining 25 flaps developed necrosis within 24 hours. The other 22 flaps remained viable until the rat was sacrificed at 7 days. The survival rate of the thigh flap was thus 88 percent. The model is suitable for use in metabolic, vascular, and immunologic studies of composite free flaps.     

rhBMP-2诱导异位软骨修复重建兔气管缺损的实验研究

目的：探讨重组人骨形成蛋白-2（rhBMP-2）作为激活物诱导异位软骨修复并重建免气管缺损的可行性。方法：取24只新西兰大白兔,制备气管前壁软骨1／3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果：术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论：rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管纽织缺损。     

The bipedicled osteocutaneous scapula flap: a new subscapular system free flap

Thirty-six adult dissections (14 cadaver and 22 operative) demonstrate the constant presence of the angular branch of the thoracodorsal artery as a vascular pedicle to the inferior pole of the scapula. This vessel originated in all cases just proximal or distal to the serratus branch of the thoracodorsal artery and arborized to the periosteum 6 to 9 cm from the bony branch of the circumflex scapular artery. In eight patients, scapular osteocutaneous flaps were raised preserving the angular branch and the circumflex scapular artery and dissecting up to the subscapular vessels. In all cases, bone was independently perfused by the angular branch. In all six cases where the angular branch was the sole supply to bone, technetium-99m scans demonstrated perfusion. Addition of this vascular pedicle to scapula bone allows two separate bone flaps with one microanastomosis and provides a longer arc of rotation between skin supplied by the circumflex scapular artery and bone. Donor-site morbidity was no greater than with the standard scapula flap.     

The simple and effective choice for treatment of chronic calcaneal osteomyelitis: neurocutaneous flaps

The authors describe their experience with the use of distally based saphenous and sural neurofasciocutaneous flaps for the treatment of calcaneal osteomyelitis in nine cases. Aggressive débridement of all nonviable and poorly vascularized tissue and coverage with a distally based neurofasciocutaneous flap were coupled with a thorough antibiotic course in all cases. The deepithelized peripheral parts of all flaps were buried in the bone cavities after bone débridement. Follow-up periods ranged from 15 to 27 months. All flaps survived completely. All of the wounds except one healed completely. These flaps have adequate blood flow for the management of chronic bone infections. They also have many advantages, such as easy quick elevation, short operative time, and acceptable donor-site morbidity. Moreover, patients treated with neurocutaneous flaps do not require debulking procedures or special shoes. Reconstruction with neurocutaneous flaps after radical débridement is a versatile alternative to the use of local or distant muscle flaps and calcanectomy procedures for patients with osteomyelitis of the os calcis.     

The role of the large superficial vein in survival of proximally based versus distally based sural veno-neuro-fasciocutaneous flaps in a rabbit model

A sural veno-neuro-fasciocutaneous flap in the New Zealand White rabbit was developed, and the role of the large subcutaneous lesser saphenous vein was investigated in proximally based versus distally based flaps. Retrograde dye injection showed that the lesser saphenous vein in rabbits has many valves with strong resistance against reflux. Twenty rabbits were randomly allocated into four groups of 10 flaps each. Group I consisted of proximally based flaps with the lesser saphenous vein intact (outflow) in the veno-neuro-adipofascial pedicle. Group II also consisted of proximally based flaps but the lesser saphenous vein was ligated at 1 cm proximal to the pedicle. Group III consisted of distally based flaps with the lesser saphenous vein intact (inflow) in the veno-neuro-adipofascial pedicle. Group IV also consisted of distally based flaps, but the lesser saphenous vein was ligated at 1 cm distal to the pedicle. The results showed that the mean flap survival area in group I (88.8 percent) was statistically higher than that in group II (62.6 percent, p < 0.001), and was higher in group IV (55.5 percent) than in group III (22.7 percent, p < 0.01). However, group II and group IV had no significant difference (p > 0.05). This experiment demonstrated that flap viability is determined by its intrinsic vascularization, both arterial and venous. The large superficial subcutaneous vein has a positive role (venous outflow) in proximally based flaps but a negative role (venous inflow) in distally based flaps. If the effect of the large subcutaneous vein is excluded, distally based flaps are not inherently inferior to proximally based flaps.     

Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate

The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved.Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively.Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.     

Revascularized periosteal grafts--a new method to produce functional new bone without bone grafting

Rib periosteum was transplanted to the groins of 9 dogs. In half of the periosteal grafts, no microvascular anastomoses were done (free grafts); at 6 weeks after grafting they had become resorbed. The other periosteal grafts were revascularized by microvascular anastomoses of the intercostal vessels to local muscular vessels; at 6 weeks those with confirmed vascular patency had all formed substantial amounts of new bone. Five cm, full-thickness defects were created in the tibias of 10 dogs. The control animals (without grafting) did not heal in two months. However, the experimental dogs, with vascularized periosteal grafts in the defects regenerated their tibias with healthy new bone by 6 weeks--and were walking on them then.     

Engineering of pre‐vascularized urethral patch with muscle flaps and hypoxia‐activated hUCMSCs improves its therapeutic outcome

Tissue engineering has brought new hopes for urethral reconstruction. However, the absence of pre‐vascularization and the subsequent degradation of materials often lead to the failure of in vivo application. In this study, with the assistance of hypoxia‐activated human umbilical cord mesenchymal stem cells (hUCMSCs), pedicled muscle flaps were used as materials and pre‐incubated in ventral penile subcutaneous cavity of rabbit for 3 weeks to prepare a pre‐vascularized urethral construct. We found that small vessels and muscle fibres were scattered in the construct after 3 weeks' pre‐incubation. The construct presented a fibrous reticular structure, which was similar to that of the corpus spongiosum under microscope examination. The produced constructs were then used as a patch graft for reconstruction of the defective rabbit urethra (experimental group), natural muscular patch was used as control (control group). Twelve weeks after the reconstructive surgery, urethrography and urethroscope inspections showed wide calibres of the reconstructed urethra in the experimental group. Histopathological studies revealed that fibrous connective tissues and abundant muscle fibres constituted the main body of the patch‐grafted urethra. In contrast, in the control group, only adipose tissue was found in the stenosis‐reconstructed urethra, replacing the originally grafted muscular tissue. To our knowledge, this is the first report that successfully constructed a pre‐vascularized urethral construct by using hypoxia‐activated hUCMSC and pedicled muscle flaps. More importantly, the pre‐vascularized construct showed a good performance in urethral reconstruction when applied in vivo. The study provided a novel strategy for tissue engineering of pre‐vascularized urethral construct for the defective urethra, representing a further advancement in urethral reconstruction.     

Surgical augmentation of skin blood flow and viability in a pig musculocutaneous flap model

A porcine rectus abdominis musculocutaneous (TRAM) flap model was designed and validated in nine pigs. This TRAM flap was based on the deep inferior epigastric (DIE) vessels with an 8 x 18 cm transverse skin paddle at the superior end of the rectus abdominis muscle. The model was subsequently used to test our hypothesis of surgical augmentation of flap viability by vascular territory expansion. Specifically, we observed that ligation of the superior epigastric (SE) vessels at 4, 7, 14, and 28 days (N = 6 to 8) prior to raising the TRAM flaps significantly increased (p less than 0.05) the length and area of the viable skin in the transverse skin paddles of the treatment flaps compared with the contralateral shammanipulated control flaps. This significant increase in skin viability was seen to be accompanied by a significant increase (p less than 0.05) in skin and muscle capillary blood flow in the treatment TRAM flaps compared with the controls (N = 9). The mechanism of vascular territory expansion is unclear. We postulate that hypoxia resulting from the ligation of the superior epigastric vessels prior to the flap surgery may play a role in the triggering of the deep inferior epigastric artery to take over some of the territory previously perfused by the superior epigastric artery. This would then increase the skin and muscle capillary blood flow in the transverse paddle when the TRAM flap was raised on the deep inferior epigastric vascular pedicle.