Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein.

BACKGROUND: The purpose of this study was to evaluate the osseous healing of a critical-sized femoral defect in a rat model with use of recombinant human bone morphogenetic protein-2 (rhBMP-2), a matrix fabricated of D,D-L,L-polylactic and hyaluronan acid (OPLA-HY), and a vascularized periosteal flap. METHODS: The carrier matrix OPLA-HY with or without rhBMP-2 was implanted in a 1-cm-long femoral defect and secured with a plate and screws. In some groups, a vascularized periosteal flap was harvested from the medial surface of the tibia. In group 1, the femoral defects in the animals were filled with the OPLA-HY matrix alone; in group 2, the OPLA-HY matrix was covered by the vascularized periosteal flap; in group 3, 20 mug of rhBMP-2 was added to the OPLA-HY matrix; and in group 4, the femoral defect containing the OPLA-HY matrix and 20 mug of rhBMP-2 was wrapped circumferentially by the vascularized periosteal flap. The presence and density of new bone formation in the femoral defect were evaluated radiographically, histologically, and with histomorphometry at four and eight weeks postoperatively. RESULTS: Groups 1 and 2, which were not treated with rhBMP-2, showed no radiographic or histologic evidence of mature bone formation at four or eight weeks. Both groups 3 and 4, which were treated with rhBMP-2, demonstrated excellent bone formation. However, with the periosteal flap, group 4 demonstrated more bone formation on histomorphometric analysis at eight weeks (43.1%) than did group 3 (28.3%) (p < 0.01). Additionally, heterotopic bone formed outside the boundaries of the defect in eight of the fifteen animals in group 3, which had no periosteal flap. CONCLUSIONS: Bone-tissue engineering with use of the OPLA-HY matrix and rhBMP-2 produced good bone formation in the rat femoral defect model. However, the addition of a vascularized periosteal flap significantly increased bone formation within the boundaries of the defect and prevented heterotopic ossification.

A rationale for delivery of osteoinductive proteins.

Highly pure, recombinant human osteoinductive proteins make it possible to consider programmable osteoneogenesis. Until recently, it was believed that a bioresorbable excipient or physiologic solution would suffice to transport osteoinductive agents from source to wound. After considering surgical requirements, particular bone wound circumstances, scarcity of collateral circulation, phenotype plasticity of mesenchymal progenitor cells, and the morphogens' pleiotrophic effects, it becomes clear that the issue of controlled, programmable osteoneogenesis is a more complicated proposition than can be addressed solely by application of osteoinductive protein. The essential characteristics of a manufactured bone graft substitute (BGS) device are dictated by demands placed on such a device by the surgeons who will employ them and the cells that will occupy them. This review outlines a design process for BGS devices that (1) begins by surveying BGS requirements gathered from the literature from 1991 to 1995, (2) briefly reviews recent in vitro studies of rhBMP-2 and OP- 1, (3) describes commonly encountered circumstances of recipient wound beds, (4) describes behaviors of mesenchymal cells involved in connective tissue repair and regeneration, and (5) concludes with a rationale for design of an osteoinductive bone graft substitute. Emerging from this process is a composite device consisting of a bioresorbable structural polymer, a filamentous velour of hyaluronan (HY), and an osteoinductive protein. The structural polymer, D,D-L,L-polylactic acid, fabricated in the architecture of cancellous bone, is capable of maintaining its structural and architectural properties after being thoroughly saturated with water. Within its interstices is located a filamentous velour of hyaluronan which, when fully hydrated, becomes a viscoelastic gel. It is anticipated that the osteoinductive protein will either be carried on the dried hyaluronic acid velour or in solution via the viscoelastic HY gel.

Practical illustrations in tissue engineering: surgical considerations relevant to the implantation of osteoinductive devices.

This paper provides practical illustrations in the use of osteoinductive devices (biomaterial carriers coupled with osteoinductive morphogens) for bone tissue engineering. We discuss the considerations relative to the implantation of these devices that may induce tissues that are located outside the boundaries of the osteoinductive device as well as outside boundaries of the normal skeletal envelope. Five reports of osteoinductive devices generating such tissues are reviewed. Histologic and radiographic data from a sixth example are presented and compared with histologic and radiographic findings typical of two varieties of myositis ossificans. A theory is advanced that osteoinductive implants may induce ectopic tissues that resemble fibro-osseous pathologies. Finally characteristics of tissue-engineered bone graft substitutes that may contribute to development of these pathologies and device characteristics that may obviate these ectopic tissues are considered.

Effects of cis-diamminedichloroplatinum II released from D,L-polylactic acid implanted adjacent to cortical allografts in dogs.

This study was performed to determine the pharmacokinetics and local and systemic effects of cis-diamminedichloroplatinum II (cisplatin) released from an open-cell polylactic acid polymer when the drug delivery device was placed adjacent to a cortical allograft. Bilateral intercalary femoral allografts were implanted in six normal beagles. The polymer containing cisplatin was implanted adjacent to the allograft in one femur, and the polymer without cisplatin was implanted adjacent to the allograft in the contralateral femur. Systemic toxicity was evaluated clinically by hematologic and serum biochemistry tests and urinalysis. Healing of the allograft was monitored radiographically. The femora were evaluated biomechanically, histologically, and histomorphometrically 7.5 months after surgery. Total serum platinum levels were measured by atomic absorption spectrophotometry, and pharmacokinetic parameters were calculated. Healing was impaired slightly by the presence of the polymer with cisplatin, and systemic and local toxicity was mild and transient. After implantation of the polymer with cisplatin, the mean peak total serum platinum concentration was low (1.71 +/- 0.19 micrograms/ml). However, the area under the curve for total serum platinum concentration versus time for the first 21 days was large (27,050 +/- 3,201 micrograms.min/ml). When cisplatin was given as an intravenous bolus at a dose of 70 mg/m2 to six other beagles, the mean peak total platinum concentration was 8.80 +/- 2.1 micrograms/ml and the area under the curve was 940.3 +/- 256.7 micrograms.min/ml. These results indicate that a sustained release of cisplatin can be delivered safely from an open-cell polylactic acid polymer. This device may be useful in the treatment of solid tumors.

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.

Effects of a controlled-release cisplatin delivery system used after resection of mammary carcinoma in mice.

OBJECTIVE: To evaluate efficacy of a controlled-release cisplatin delivery system, used after marginal resection of mammary carcinoma (ie, resection of grossly evident tumor) in mice, to prevent tumor regrowth and metastasis. ANIMALS: 42 female C3H-HeJ mice. PROCEDURE: Mice were inoculated with mammary carcinoma cells. Between 2 and 6 days later, tumors were marginally resected and mice were assigned to 1 of 3 groups: no treatment (control; n = 14), cisplatin administered intraperitoneally (i.p. cisplatin; 14), and cisplatin delivered by use of an open-cell polylactic acid system placed within the tumor bed (slow-release cisplatin; 14). Tumor regrowth was measured daily. Mice were euthanatized 14 days after surgery, and complete necropsies were performed. RESULTS: Tumor regrowth was not detected in the slow-release cisplatin group; however, tumor regrowth was detected in 7 of 14 mice in the i.p. cisplatin group and 14 of 14 mice in the control group. Median (+/-SD) number of days to tumor regrowth was 13.5+/-0.64 and 7.79+/-0.87 in the i.p. cisplatin and control groups, respectively. Mice in the i.p. cisplatin group had significantly delayed tumor regrowth, compared with control mice. Metastases to lungs were detected in 8 of 14 control mice but were not detected in mice in either cisplatin treatment group. CONCLUSIONS AND CLINICAL RELEVANCE: The open-cell polylactic acid with cisplatin delivery system was successful in delaying local tumor regrowth and metastasis in mice with marginally resected mammary carcinoma. Use of a controlled-release cisplatin delivery system may be an effective adjunct treatment following excision of mammary carcinoma in humans and other animals.

Regeneration of articular cartilage--evaluation of osteochondral defect repair in the rabbit using multiphasic implants.

OBJECTIVE: To investigate whether two different multiphasic implants could initiate and sustain repair of osteochondral defects in rabbits. The implants address the malleable properties of cartilage while also addressing the rigid characteristics of subchondral bone. DESIGN: The bone region of both devices consisted of D, D-L, L-polylactic acid invested with hyaluronan (HY). The cartilage region of the first device was a polyelectrolytic complex (PEC) hydrogel of HY and chitosan. In the second device the cartilage region consisted of type I collagen scaffold. Eighteen rabbits were implanted bilaterally with a device, or underwent defect creation with no implant. At 24 weeks, regenerated tissues were evaluated grossly, histologically and via immunostaining for type II collagen. RESULTS: PEC devices induced a significantly better repair than untreated shams. Collagen devices resulted in a quality of repair close to that of the PEC group, although its mean repair score (19.0+/-4.2) did not differ significantly from that of the PEC group (20.4+/-3.7) or the shams (16.5+/-6.3). The percentage of hyaline-appearing cartilage in the repair was highest with collagen implants, while the degree of bonding of repair to the host, structural integrity of the neocartilage, and reconstitution of the subchondral bone was greatest with PEC devices. Cartilage in both device-treated sites stained positive for type II collagen and GAG. CONCLUSIONS: Both implants are capable of maintaining hyaline-appearing tissue at 24 weeks. The physicochemical region between the cartilage and bone compartments makes these devices well suited for delivery of different growth factors or drugs in each compartment, or different doses of the same factor. It also renders these devices excellent vehicles for chondrocyte or stem cell transplantation.

Melanotic neuroectodermal tumor of infancy.

Recurrence of a melanotic neuroectodermal tumor of infancy was clinically evident eight weeks after the original lesion had been surgically removed. Concentrations of urinary vanilmandelic acid were elevated immediately after surgery but returned to normal a month after removal of the tumor. The location of the recurrent tumor, the rapidity of recurrence, and the concentrations of urinary VMA favor a theory of neural crest origin.

Foreign body giant cell reaction related to placement of tetracycline-treated polylactic acid: report of 18 cases.

Presented are 18 cases of foreign body giant cell reaction in mandibular third molar extraction wounds previously treated with 40 mg of dry tetracycline powder carried into the wound with a biodegradable polymer dressing. The lesions vary in intensity from local, soft tissue granulomas to osteolytic, central bone pathologies. Several reports of foreign body giant cell reaction to insoluble tetracycline preparations are cited, along with a brief review of the relationship between myospherulosis and various formulations of tetracycline powder in petroleum-based carrier vehicles. The authors suggest that the foreign body giant cell lesions reported here were initiated by micron-sized particles of insoluble tetracycline powder and were further aggravated by certain hydrophobic characteristics of the associated biodegradeable polymer. The authors caution against use of dry, powdered forms of topical antibiotics in fresh dental extraction wounds.

Principles of tissue engineering applied to programmable osteogenesis.

This article presents a strategy for design, engineering, and fabrication of a bioresorbable, manufactured bone graft substitute (BGS) device. The approach is based on established precepts of osteogenesis, molecular biology of hyaluronic acid and osteoinductive proteins, and theoretical preformance criteria for such a device collated from the literature of 1991 to 1996. Application of this design and engineering strategy results in a composite device consisting of a D,D-L,L-polylactic acid macrostructure optimized to the architecture of cancellous bone, a microstructure composed of a filamentous velour of hyaluronan and a recombinant human bone morphogenetic protein 2 (rhBMP-2). The performance of this construct was tested in vivo in the dog, intertransverse process, spinal fusion model and in a critical sized defect of the rabbit radius. Data from these studies are used to illustrate principle points of the design and engineering concept.

[Heterotopic and orthotopic bone formation with a vascularized periosteal flap, a matrix and rh-BMP-2 (bone morphogenetic protein) in the rat model]. / Heterotope und orthotope Knochenbildung mit einem vaskularisierten Periostlappen, einer Matrix und rh-BMP-2 (bone morphogenetic protein) im Rattenmodell.

The purpose of this study was to construct a vascularized bone graft using the osteoinductive bone morphogenetic protein (rh-BMP-2), a polylactic acid matrix (OPLA/HY), and a vascularized periosteal flap containing osteoprogenitor cells ectopically in the groin or orthotopically in a femoral defect. In the Lewis rat, periosteal flaps were harvested from the medial surface of the tibia vascularized by the saphenous artery and vein and were transferred to the groin on its vascularized pedicle. Alternatively, the periosteal flap along its pedicle was transferred between the thigh muscles to be wrapped around a femoral defect of 1 cm. The animals were divided into 10 groups (82 animals). In group 1, the periosteal flap was left empty in the groin. Groups 2 and 3 consisted of the periosteal flap and 20 micrograms rh-BMP-2, but in group 3 the vascular pedicle was ligated proximally. In group 4 the flap was harvested without the periosteal layer and turned "inside out". Groups 5 and 6 consisted of the periosteal flap and the matrix OPLA/HY +/- 20 micrograms rh-BMP-2. In the femoral defect model, bone formation was studied using the matrix OPLA/HY alone (group 7) or combined with the vascularized periosteal flap (group 8), or in combination with OPLA/HY + BMP (group 9) or OPLA/HY + BMP + the periosteal flap (group 10). The presence and density of new bone formation in the groin and femoral defect were evaluated radiologically and histologically at 4 and 8 weeks. Good bone formation in the groin chamber (ectopic) was demonstrated in the periosteal flap + OPLA/HY + BMP group. In the femoral defects, good bone formation (orthotopic) was seen in the OPLA/HY + BMP + the periosteal flap groups. However, with the presence of a vascularized periosteal flap, more bone formation along the rim of the defect was observed. This study of ectopic bone formation in the groin and orthotopic bone formation in the femoral defect demonstrates that optimal bone formation requires four factors: BMP, a biodegradable matrix, osteoprogenitor cells, and blood supply. Potentially in the future, this technique could be used to reconstruct a bony defect or a nonunion by covering the involved area with a vascularized periosteal flap and a suitable matrix combined with BMP. Alternatively, a vascularized bone graft could be prefabricated at a distant site and then transferred microsurgically into a defect.

Influence of polylactic acid mesh on the incidence of localized osteitis.

A biodegradable polylactic acid surgical implant, designed to facilitate dental extraction wound heading, is described. Animal studies have proved the safety and tissue compatibility of the implanted polylactic acid material. A clinical study using this material in mandibular third molar extraction sites in human subjects is reviewed. Results of that study reveal the implant to be effective in reducing the incidence of localized osteitis following mandibular third molar extractions.

Slow release cisplatin combined with radiation for the treatment of canine nasal tumors.

Thirteen dogs with malignant tumors of the nasal cavity were treated with a combination of slow release cisplatin and megavoltage radiation. Radiation was delivered on a Monday through Friday schedule using a 6 MV linear accelerator. The median total dose was 49.5 Gy (range 49.5-56 Gy). Cisplatin was given using an open-cell polylactic acid polymer, impregnated with the drug and implanted intramuscularly at a distant site, as a slow release delivery system (OPLA-Pt [THM Biomedical, Inc]). The median dose used was 60 mg/m2 (range 60-100 mg/m2). When combined with radiation, this delivery system caused no systemic drug toxicity, and a local tissue reaction was seen in only two dogs. Acute side effects to normal tissue from radiation were not enhanced, as measured by subjective assessment. When compared to a group of historical controls that received radiation without OPLA-Pt, the dogs that received combined radiation and cisplatin had longer overall survival times, with a median of 580 days. The control group had a median survival of 325 days. Previously reported median survival times for comparable megavoltage radiation treatment range from 6 to 13 months. Some dogs in both groups also received adjubant chemotherapy but this did not influence survival time. By multivariate analysis, only the use of OPLA-Pt was found to significantly influence survival, with a p value of p = 0.023. Mega-voltage radiation and slow release cisplatin appears to be a well tolerated combination that may favorably affect survival of dogs with nasal tumors.

Radiomorphometry and biomechanical assessment of recombinant human bone morphogenetic protein 2 and polymer in rabbit radius ostectomy model.

The study objective was to determine the mechanical integrity and radiopacity of regenerated bone within critical-sized defects (CSDs) in radii of rabbits using recombinant human bone morphogenetic protein 2 (rhBMP-2) with a porous, biodegradable poly(D,L-lactic acid) (PDLLA) carrier (designated PLA). Twenty millimeter, unilateral radial ostectomies were created in 96 skeletally mature New Zealand white rabbits. The rabbits were randomly assigned to six treatment groups with two euthanasia periods. Treatment groups included unfilled defect (n = 8), segmental autograft (n = 8), PLA + 0 microg rhBMP-2 (n = 8), PLA + 17 microg rhBMP-2 (n = 8), PLA + 35 microg rhBMP-2 (n = 8), and PLA + 70 microg rhBMP-2 (n = 8). The radiopacity was significantly greater for the 35- and 70-microg rhBMP-2 groups at 4 weeks compared to unfilled controls, PLA only, and 17-microg rhBMP-2 groups and equivalent to the autograft. At 8 weeks all groups receiving rhBMP-2 were equivalent to the autograft and significantly greater than unfilled defects and PLA alone. Similarly, the biomechanical analysis indicated significantly greater torque at failure for the 35-microg rhBMP-2 group compared to all other groups at 4 weeks. By 8 weeks all groups receiving rhBMP-2 and autograft had significantly greater torque than unfilled controls and PLA alone. These radiomorphometric and biomechanical results indicate PLA may be a suitable carrier for rhBMP-2 used for skeletal regeneration.