Evaluation of osteogenic/chondrogenic cellular proliferation and differentiation in the xenogeneic periosteal graft

To determine whether grafted young periosteum can induce new bone formation in elderly patients, this preliminary study evaluated cell proliferation and differentiation in xenogeneic periosteal grafts in old rats radiographically, histologically, and immunohistochemically. Periosteum harvested from the tibia of young Japanese white rabbits were grafted into old Sprague-Dawley rats with or without administration of 1.0 mg per kilogram per day immunosuppressant FK506. Autogenous old periosteal tissue grafts were also evaluated as a control. Grafted tissue was extirpated after 7, 14, 21, and 45 days. In the xenogeneic group, proliferative cell nuclear antigen-positive cells were observed 7 days after surgery, which differentiated into chondroblasts with bone morphogenetic protein-2 expression and finally formed cartilage by 14 days. Endochondral ossification was observed at 21 days, and bone replacement was completed by 45 days. No osteogenic cell activity was observed in the two other groups. Xenogeneic young periosteum thus maintained its osteogenic/chondrogenic potentiality in older rats.     

Free calvarial periosteum graft vascularized by an omental flap in a rat model

Because omental flaps are useful for flap prefabrication and the cambium layer of the periosteum can be osteogenic, we examined whether calvarial periosteum grafted onto greater omentum of rats was osteogenic and suitable for a flap. Distal omentum was wrapped with calvarial periosteum and so the cambium faced the omentum. Grafted omentum was harvested at 1 to 9 days. In other rats, grafted omentum was elevated as a pedicled flap and moved to the abdominal subcutis, to be harvested later at 1 to 5 months after the initial surgery. Bone formation was evaluated histologically, histochemically, and radiographically. On day 3, osteoid had formed. From day 4, calvarial periosteum was revascularized by omentum and bone was forming. New bone was maintained after grafting to subcutis for 5 months. Thus, bone formed by periosteum on the omentum could be used to reconstruct defects of the bone.     

Immunolocalization of vascular endothelial growth factor during heterotopic bone formation induced from grafted periosteum

Vessel invasion is an important step in cartilage replacement that leads to bone formation, and vascular endothelial growth factor (VEGF) has been implicated as a key player in this process. Although grafted periosteum undergoes endochondral ossification, little is known about the role of VEGF in this process. In the current study the authors investigated by immunohistochemical, histochemical, and ultrastructural techniques the localization of VEGF during bone formation in periosteal grafts. At day 14 after grafting the tibias of Japanese white rabbits, periosteal cells in the grafted tissue had differentiated into chondrocytes to form cartilage. Some chondrocytes were immunopositive for VEGF expression, and subsequent vessel invasion occurred predominantly in these VEGF-positive areas. At day 45, the cartilage invaded by blood vessels had been replaced by newly formed bone. These findings suggest that VEGF is associated with the process of blood vessel invasion into cartilage before bone replacement in endochondral ossification from grafted periosteum.     

Osteogenic potential of primed periosteum graft in the rat calvarial model

Repair of bone defects remains a major concern in plastic and maxillofacial surgery. Based on modern concepts of tissue engineering, periosteum has gained attention as a suitable osteogenic material. We tested the hypothesis that surgically released and immediately repositioned periosteum would exhibit high osteogenic capacity upon grafting in a rat calvarial defect. Seven days after periosteum was released from the tibia and immediately repositioned, the "primed periosteum graft" (PPG; n = 15) was placed into a critical-sized defect of rat calvaria and the process of bone formation was evaluated histologically, immunohistologically, and radiographically at 7, 14, and 21 days after grafting. Findings were compared with a nonprimed periosteal graft (NPG; n = 15).Endochondral ossification was observed in both the PPG and NPG. The PPG showed higher expression of proliferative cell nuclear antigen, bone morphogenetic protein, and vascular endothelial growth factor than the NPG. Three-dimensional radiographic examination revealed significantly increased bone formation in the PPG than in the NPG (P < 0.01). These findings suggested that surgical stimulation of the periosteum enhanced the osteogenic potential of periosteal cells. This method may be suitable for the clinical repair of bone defects.     

Role of fracture hematoma and periosteum during fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step of the healing process

To study the mechanisms of fracture healing, we investigated the interaction between fracture hematoma and periosteum during the early phase of fracture healing in rats. Experimentally induced fractures of the tibia in untreated rats were compared histologically with such fractures in rats in which either the bone marrow or the periosteum had been removed. The extent of periosteal cell proliferation and chondrogenesis in the fracture hematoma was evaluated on experimental days 3, 6, 10, and 14. On day 3, periosteal cell proliferation at the tibial fracture site was decreased in the bone marrow-removed rats compared with the proliferation in untreated rats. Little chondrogenesis in the fracture hematoma was seen through day 6 in the periosteum-removed rats. These results suggest that the periosteum is important for mediating the primary steps of chondrogenesis and enchondral ossification in the fracture hematoma and that the fracture hematoma may be essential for periosteal cell proliferation during fracture healing. Received for publication on April 5, 1999; accepted on July 21, 1999     

The evaluation of bone formation of the whole-tissue periosteum transplantation in combination with beta-tricalcium phosphate (TCP)

We investigated the osteogenic potential of a combination graft of beta-tricalcium phosphate (TCP) and periosteum in the rat calvarial defect model. The combination beta-TCP and periosteum graft was grafted into rat calvarial defects; the newly formed bone in the defect was studied histologically and radiographically and compared with periosteum grafts and TCP grafts. Ten days after combination grafting, the grafted periosteum showed cell proliferation and Runx2 immunoreaction; 20 days after grafting, new bone formation was seen around the beta-TCP; and 30 days after grafting, new bone developed and actively replaced beta-TCP, while radiography showed calcified areas. Total bone formation of the combination periosteum and beta-TCP graft was significantly increased compared with single grafts of beta-TCP or periosteum (P < 0.01). The combination graft of periosteum and beta-TCP showed marked bone formation in rat calvarial defects. This result suggests that combination grafts may be effective for repairing bone defects.     

Vascular proliferation and blood supply during distraction osteogenesis: a scanning electron microscopic observation.

This scanning electron microscopic study examined the spatial and temporal features of proliferating vessels of regenerating bone tissue and blood supply during distraction osteogenesis. A rat model of tibial lengthening was used with a protocol divided into a latency period of 7 days, a distraction period that lasted 14 days with a daily distraction rate of 0.5 mm in two steps, and a consolidation period of 21 days. Vascular casting was done on the hindlimbs before osteotomy and on postoperative days 7, 14, 21, 28, and 42. Scanning electron microscopic findings were correlated with radiological and histological observations. On days 7 and 14, the proliferation of periosteal vessels was pronounced and there was distinct subperiosteal bone formation on the osteotomized surfaces. On day 21, vascular branches from the medullary canal of the host bone formed a vascular network, which gave rise to multiple axial, straight vascular branches, running parallel to the direction of distraction, toward the interzone, in accordance with the progress of mineralization. On day 28, the periosteum provided vascularization to the peripheral side of the interzone whereas the center of the interzone was still relatively avascular. On day 42, the periosteal and medullary vascular channels were completely connected at the distraction site including the interzone, which was occupied by developing and mature bone trabeculae. These results suggest that vascular proliferation occurs actively during the latency and distraction periods and then gradually decreases over time. A close temporal and spatial relationship exists between formation of regenerated bone and vascular proliferation of the periosteum and medullary canal.     

Comparative study of technetium-99m bone scans and orthopantomography in determining mandible invasion in intraoral squamous cell carcinoma

The accuracy of preoperative assessment in determining invasion of the mandible by intraoral squamous cell carcinoma was analyzed in 48 patients who underwent mandibulectomy, and the results correlated with the histopathological reports of the resected specimens. Only 50% of the patients underwent the "ideal" surgery based primarily on clinical judgement, whereas 10 patients in the series were significantly undertreated. Clinical judgement and routine preoperative x-rays are accurate in cases where there is gross involvement of the mandible (17 of 19) but are significantly less successful in determining early bone invasion, invasion of the periosteum, or periosteal new bone formation. In such cases (26 of 48), a technetium-99m bone scan provides additional information. A grading system for reporting orthopantomographics (OPTs) and bone scans has been developed and utilized to form a reference grid to determine the optimum extent of mandibular surgery. The results show that using this protocol, unnecessary mandibular surgery may be reduced and inadequate surgical excision avoided.     

Bone formation by vascularized periosteal and osteoperiosteal grafts

The osteogenic capacity of vascularized periosteal and osteoperiosteal grafts was investigated in 82 Wistar rats about 8 weeks old. The periosteal flaps, pedicled on the descending genicular artery, were taken by stripping the lower third of the femur. In the right hindleg, the grafts were made with periosteum only, while in the left hindleg, the periosteal flaps were associated with cancellous bone. The animals were divided into two groups of 41. In group I, both the periosteal and osteoperiosteal grafts were placed in contact with cortical bone, and in group II, the grafts were buried in muscle. Subgroups of 8 animals were killed after 1, 2, 4, 8, and 16 weeks postoperatively. The grafted region was evaluated radiographically, macroscopically, and histologically. Membranous ossification was the main source of bone formation. Osteoperiosteal grafts produced a greater amount of new bone than periosteal ones. There was evidence that the contact of the graft with living cortical bone favored bone formation.     

Effects of osteocytes on osteoinduction in the autogenous rib graft in the rat mandible

In order to clarify the influence of cell death of osteocytes on osteoinduction after bone grafting, autogenous fresh ribs, bone-marrow-removed fresh ribs, and frozen devitalized ribs were grafted after removal of the periosteum in a bridge manner in the rat mandible, and the process of bone remodeling was studied histologically, histochemically, and ultrastructurally in the central portion of the grafts. In the fresh bone group, osteocytes maintained normal morphology and grafted bones were undergoing resorption by osteoclasts with ruffled borders and strong tartrate-resistant acid phosphatase (TRACP) activity on the fifth day (Day 5). Alkalinephosphatase (ALP)-positive osteoblast-like cells were observed in close proximity of the osteoclasts. On Days 7 to 9, new bone formation occasionally accompanied by newly formed cartilage was observed in the grafted bones, and by Day 14, the majority of the grafted bones had been replaced by newly formed bone. In the marrow-removed fresh bone group, bone resorption by TRACP-positive cells and new bone formation similar to those seen in the fresh bone group were observed on Day 10, In the frozen devitalized bone group in which osteocytes had undergone necrosis, bone resorption and new bone formation were not observed even on Day 84, and grafted bones became surrounded by fibrous tissues. The TRACP activity was very weak and no ruffled border was observed ultrastructurally in multinucleated giant cells seen on Day 14. In conclusion, immediate bone resorption by osteocytes is essential for osteoinduction in the bone graft, and living osteocytes in the graft play an important roll in the differentiation and activation of osteocytes.     

Histological study of effect of bone morphogenetic protein derived from bovine tooth on periosteum in rats

Summary In this study, we examined histologically the effect of a bone morphogenetic protein (BMP) derived from bovine tooth on the periosteum. Supraperiosteal injection of crude BMP into femurs of Wistar rats (28 day old) resulted in periosteal cell proliferation with subsequent bone and cartilage formation. Moreover, proliferating periosteal cells migrated into injected BMP, and formed both cartilage and bone. These observations show that exogenous BMP stimulates mesenchymal cells of the periosteum to proliferate and differentiate into osteoblasts, and therefore BMP may be one of factors which are involved in differentiation of osteoblasts in the periosteum.     

Different sympathetic pathways control the metabolism of distinct bone envelopes

Bone remodeling, the mechanism that modulates bone mass adaptation, is controlled by the sympathetic nervous system through the catecholaminergic pathway. However, resorption in the mandible periosteum envelope is associated with cholinergic Vasoactive Intestinal Peptide (VIP)-positive nerve fibers sensitive to sympathetic neurotoxics, suggesting that different sympathetic pathways may control distinct bone envelopes. In this study, we assessed the role of distinct sympathetic pathways on rat femur and mandible envelopes. To this goal, adult male Wistar rats were chemically sympathectomized or treated with agonists/antagonists of the catecholaminergic and cholinergic pathways; femora and mandibles were sampled. Histomorphometric analysis showed that sympathectomy decreased the number of preosteoclasts and RANKL-expressing osteoblasts in mandible periosteum but had no effect on femur trabecular bone. In contrast, pharmacological stimulation or repression of the catecholaminergic cell receptors impacted the femur trabecular bone and mandible endosteal retromolar zone. VIP treatment of sympathectomized rats rescued the disturbances of the mandible periosteum and alveolar wall whereas the cholinergic pathway had no effect on the catecholaminergic-dependent envelopes. We also found that VIP receptor-1 was weakly expressed in periosteal osteoblasts in the mandible and was increased by VIP treatment, whereas osteoblasts of the retromolar envelope that was innervated only by tyrosine hydroxylase-immunoreactive fibers, constitutively expressed beta-2 adrenergic receptors. These data highlight the complexity of the sympathetic control of bone metabolism. Both the embryological origin of the bone (endochondral for the femur, membranous for the mandibular periosteum and the socket wall) and environmental factors specific to the innervated envelope may influence the phenotype of the sympathetic innervation. We suggest that an origin-dependent imprint of bone cells through osteoblast-nerve interactions determines the type of autonomous system innervating a particular bone envelope.     

Articular cartilage defects in a rabbit model, retention rate of periosteal flap cover

BACKGROUND: A periosteal flap is frequently used in procedures involving repair of articular cartilage defects. Hypertrophy of the repair tissue, probably from a retained periosteum, is a clinical problem but not much is known about this issue. The objective of the present experimental study was to investigate the retention rate of periosteal flaps with respect to various postoperative mobilization regimes and the introduction of bone marrow elements underneath the flap. METHOD: We created a chondral lesion (diameter 4 mm) in both patellas of 18 New Zealand white rabbits. The subchondral bone was left intact in one knee. In the other, the bone plate was perforated, allowing bone marrow elements to enter the defect. All defects were covered with a periosteal flap, sutured and glued to the rim of the cartilage defect. Postoperatively, the rabbits were allocated to one of three groups: A. rehabilitation in cages for 4 days, then killed; B. rehabilitation in cages for 7 days, then free activity on the floor of a 10 m2 room until the end of the second week, then killed; C. rehabilitation in cages for 2 weeks, then killed. RESULTS: 16 of 23 periosteal flaps became detached within 2 weeks (one knee was excluded because of clinical signs of infection), with no difference in the retention rate with respect to mobilization regime or established access to bone marrow elements in the defect. The periosteum still served as a cover of the defect in 10 of 12 knees at day 4. This figure decreased to 7 of 23 knees at day 14. CONCLUSION: Our study is the first to document the retention rate of periosteal flaps in a rabbit model. The low retention rate observed may explain why periosteal hypertrophy is not reported in experimental studies where the periosteal flap is sutured to the cartilage rim.     

Electrically stimulated periosteal grafting in the rat

Osteogenesis of the periosteum was examined in rats. The periosteum electrically stimulated for 6 days was grafted into the axillar muscle, and new bone tissue was found in 40 out of 44 animals (90.9%). Periosteum without stimulation and that with only 2 days of electrical stimulation did not generate new bone tissue. The periosteum electrically stimulated for 4 days did not show substantial new bone formation in the muscle. The periosteal tissue which was initially electrically stimulated for 6 days was definitely transformed into new bone tissue in the muscle. This shows that boneless bone grafting can be accomplished successfully in rats by electrically stimulating the periosteum before grafting. Periosteal grafting is considered to be potentially valuable as a boneless bone grafting technique.     

Effects of pulsed electromagnetic field stimulation on distraction osteogenesis in the rabbit tibial leg lengthening model

The purpose of this study was to determine whether exposure to pulsed electromagnetic field (PEMF) would shorten the healing time of regenerate bone in a rabbit tibial distraction model. Beginning 1 day after surgery, mid-shaft tibial osteotomies, stabilized with external fixators, were distracted 0.25 mm twice daily for 21 days and received either no exposure (sham control) or 1 hour per day exposure to low-amplitude, low-frequency PEMF. Tibiae were tested for torsional strength after 9, 16, and 23 days post-distraction. PEMF-treated tibiae were significantly stronger than shams at all three time points. By 16 days post-distraction, the PEMF group had achieved biomechanical strength essentially equivalent to intact bone. Shams did not achieve normal biomechanical strength even after 23 days post-distraction. In this tibial distraction model, short daily PEMF exposures accelerated consolidation of regenerate bone. Clinical usefulness awaits testing.     

Critical size defect in the canine mandible.

OBJECTIVE: The purpose of this study was to determine the minimum size defect in a canine mandible that would not spontaneously heal during the dog's natural life (the critical size defect). STUDY DESIGN: Sixteen adult female mongrel dogs underwent continuity resection on both sides of the mandible to create bilateral defects. In 8 dogs, mandibular defects ranging from 5 to 20 mm were created with periosteal resection. In the other 8 dogs, mandibular defects ranging from 30 to 60 mm were created preserving the periosteum. The dogs were then killed at 6 months and the defects examined using radiographs and histologic analysis. RESULTS: When the periosteum was removed, mandibular defects greater than 15 mm failed to heal across the entire defect. However, when the periosteum was preserved, mandibular defects needed to be greater than 50 mm in order to fail to heal. CONCLUSION: The critical size defect in a canine mandible model is 15 mm when the periosteum is removed and 50 mm when the periosteum is preserved.     

Testing of a new one-stage bone-transport surgical procedure exploiting the periosteum for the repair of long-bone defects

BACKGROUND: A recently proposed one-stage bone-transport surgical procedure exploits the intrinsic osteogenic potential of the periosteum while providing mechanical stability through intramedullary nailing. The objective of this study was to assess the efficacy of this technique to bridge massive long-bone defects in a single stage. METHODS: With use of an ovine femoral model, an in situ periosteal sleeve was elevated circumferentially from healthy diaphyseal bone, which was osteotomized and transported over an intramedullary nail into a 2.54-cm (1-in) critical-sized diaphyseal defect. The defect-bridging and bone-regenerating capacity of the procedure were tested in five groups of seven animals each, which were defined by the absence (Group 1; control) or presence of the periosteal sleeve alone (Group 2), bone graft within the periosteal sleeve (Groups 3 and 5), as well as retention of adherent, vascularized cortical bone chips on the periosteal sleeve with or without bone graft (Groups 4 and 5). The efficacy of the procedure was assessed qualitatively and quantitatively. RESULTS: At sixteen weeks, osseous bridging of the defect was observed in all twenty-eight experimental sheep in which the periosteal sleeve was retained; the defect persisted in the remaining seven control sheep. Among the experimental groups 2 through 5, significant differences were observed in the density of the regenerated bone tissue; the two groups in which vascularized bone chips adhered to the inner surface of the periosteal sleeve (Groups 4 and 5) showed a higher mean bone density in the defect zone (p < 0.02) than did the other groups. In these two groups with the highest bone density, the addition of bone graft was associated with a significantly lower callus density than that observed without bone graft (p < 0.05). The volume of regenerate bone (p < 0.02) was significantly greater in the groups in which the periosteal sleeve was retained than it was in the control group. Among the experimental groups (groups 2 through 5), however, with the numbers studied, no significant differences in the volume of regenerate bone could be attributed to the inclusion of bone graft within the sleeve or to vascularized bone chips remaining adherent to the periosteum. CONCLUSIONS: The novel surgical procedure was shown to be effective in bridging a critical-sized defect in an ovine femoral model. Vascularized bone chips adherent to the inner surface of the periosteal sleeve, without the addition of morselized cancellous bone graft within the sleeve, provide not only a comparable volume of regenerate bone and composite tissue (callus and bone) but also a superior density of regenerate bone compared with that after the addition of bone graft.     

Articular cartilage defects in a rabbit model,retention rate of periosteal flap cover

Background A periosteal flap is frequently used in procedures involving repair of articular cartilage defects. Hypertrophy of the repair tissue, probably from a retained periosteum, is a clinical problem but not much is known about this issue. The objective of the present experimental study was to investigate the retention rate of periosteal flaps with respect to various postoperative mobilization regimes and the introduction of bone marrow elements underneath the flap.

Method We created a chondral lesion (diameter 4 mm) in both patellas of 18 New Zealand white rabbits. The subchondral bone was left intact in one knee. In the other, the bone plate was perforated, allowing bone marrow elements to enter the defect. All defects were covered with a periosteal flap, sutured and glued to the rim of the cartilage defect. Postoperatively, the rabbits were allocated to one of three groups: A. rehabilitation in cages for 4 days, then killed; B. rehabilitation in cages for 7 days, then free activity on the floor of a 10 m2 room until the end of the second week, then killed; C. rehabilitation in cages for 2 weeks, then killed.

Results 16 of 23 periosteal flaps became detached within 2 weeks (one knee was excluded because of clinical signs of infection), with no difference in the retention rate with respect to mobilization regime or established access to bone marrow elements in the defect. The periosteum still served as a cover of the defect in 10 of 12 knees at day 4. This figure decreased to 7 of 23 knees at day 14.

Conclusion Our study is the first to document the retention rate of periosteal flaps in a rabbit model. The low retention rate observed may explain why periosteal hypertrophy is not reported in experimental studies where the periosteal flap is sutured to the cartilage rim.     

Ischemic necrosis of the femoral head: An experimental rabbit model

To describe the morphology of the proximal femoral epiphysis in a rabbit model from the ischemic insult to the end of the revascularization process. Ischemia of the femoral head was induced in 32 rabbits at the 8th day of life, by sectioning the joint capsule and the ligamentum teres and dislocating the femoral head. Rabbits were sacrificed at 4, 8, 12, 18, 21, 26, 34, and 48 days after surgery and femoral heads were observed histologically. During the first days following the ischemic injury, large areas underwent necrotic changes. Both epiphyseal and physeal cartilage were thicker than normal and less trabecular bone formation was evident. Bone marrow was also diffusely necrotic within the secondary center of ossification. After day 12th, reparative process started with formation of extensive areas of fibrocartilage and several secondary centers of ossifications. At that stage femoral head deformity was already evident. In the following days the secondary centers of ossification cohalesced and epiphyseal and physeal cartilage resumed a normal appearance, but the femur showed a permanent deformity. In newborn rabbits, the ischemic injury to the femoral head blocked the ossification of the epiphyseal and physeal cartilage associated to necrotic bone marrow within the secondary center of ossification of the femoral head as well as to extensive areas of necrosis of epiphyseal and physeal cartilage. Extensive areas of fibrocartilage and small newly formed ossification centers within the femoral epiphysis were the results of the revascularization process, and femoral head deformity became stable afterward. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:535–541, 2015.     

Subperiosteal tissue expansion: an experimental study

Periosteal flaps have been used to cover bone defects. There are no sufficient data on whether expanded periosteum can be used to repair bone defects after subperiosteal tissue expansion. In this experimental study, 14 male dogs, which were 2 years-old and weighed 14.5–16.0 kg, were used to investigate the repair of bony defects with expanded periosteum. Rectangular tissue expanders, measuring 50 mm in width, 70 mm in length, and 20 mm in height (volume: 20 ml) were used. The dogs were divided into two groups each of seven dogs: one control group and one experimental group. In each dog, a pocket, 50×70 mm in size, was prepared in parietal area, and a periosteal area, 30×40 mm in size, was marked out. A rectangular tissue expander was inserted into the pocket. In the control group, the tissue expanders were not inflated. On the seventh postoperative day, the tissue expander inflation with normal saline (5 ml per 2 days) was started in the experimental group. After 15 days, the previously marked out periosteal area was measured. A specimen was taken from this periosteal tissue for histopathological evaluation. A bone defect, 20×20 mm in size, was created beneath expanded periosteal tissue, and this defect was covered with the expanded periosteal tissue. After 15 days, a histopathological evaluation was carried out. Statistical analysis was carried out using the Mann–Whitney U-test. The area of periosteum expanded was more in the experimental group than that of the control group (p>0.05). After removal of the tissue expander, both in the control and subperiosteal tissue expansion group, osteoblastic lining of the expanded periosteum, increased vascularity, and granulation tissue was noted. Following 15 days, woven and lamellated bone tissue was formed both in the control and subperiosteal expansion group. In the subperiosteal expansion group, there was greater bone formation, the expanded periosteum was thickened, and thickened lamellated bone was covered by active osteoblasts. It is concluded that subperiosteal tissue expansion may be worthwhile to provide large periosteal flaps for the repair of large bone defects, by increasing osteogenic capacity.