Arnold S. Breitbart

tissue Engineered Bone Repair of Calvarial Defects Using Cultured Periosteal Cells

&NA; Periosteum has been demonstrated to have cell populations, including chondroprogenitor and osteoprogenitor cells, that can form both cartilage and bone under appropriate conditions. In the present study, periosteum was harvested, expanded in cell culture, and used to repair critical size calvarial defects in a rabbit model. Periosteum was isolated from New Zealand White rabbits, grown in cell culture, labeled with the thymidine analog bromodeoxyuridine for later localization, and seeded into resorbable polyglycolic acid scaffold matrices. Thirty adult New Zealand White rabbits were divided into groups, and a single 15‐mm diameter full‐thickness calvarial defect was made in each animal. In group I, defects were repaired using resorbable polyglycolic acid implants seeded with periosteal cells. In group II, defects were repaired using untreated polyglycolic acid implants. In group III, the defects were left unrepaired. Rabbits were killed at 4 and 12 weeks postoperatively. Defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype, with production of osteocalcin upon 1,25(OH)2 vitamin D3 induction. In vivo results at I weeks showed islands of bone in the defects repaired with polyglycolic acid implants with periosteal cells (group I), whereas the defects repaired with untreated polyglycolic acid implants (group II) were filled with fibrous tissue. Collagen content was significantly increased in group I compared with group II (2.90 ± 0.80 &mgr;g/mg dry weight versus 0.08 ± 0.11 &mgr;g/mg dry weight, p < 0.006), as was the ash weight (0.58 ± 0.11 mg/mg dry weight versus 0.35 ± 0.06 mg/mg dry weight, p < 0.015). At 12 weeks there were large amounts of bone in group I, whereas there were scattered islands of bone in groups II and III. Radiodensitometry demonstrated significantly increased radiodensity of the defect sites in group I, compared with groups II and III (0.740 ± 0.250 OD/mm2 versus 0.404 ± 0.100 OD/mm2 and 0.266 ± 0.150 OD/mm2, respectively, p < 0.05). Bromodeoxyuridine label, as detected by immunofluorescence, was identified in the newly formed bone in group I at both 4 and 12 weeks, confirming the contribution of the cultured periosteal cells to this bone formation. This study thus demonstrates a tissue‐engineering approach to the repair of bone defects, which may have clinical applications in craniofacial and orthopedic surgery.

Cartilage and bone regeneration using gene-enhanced tissue engineering.

Joint cartilage injury remains a major problem in orthopaedics with more than 500,000 cartilage repair procedures performed yearly in the United States at a cost of hundreds of millions of dollars. No consistently reliable means to regenerate joint cartilage currently exists. The technologies of gene therapy and tissue engineering were combined using a retroviral vector to stably introduce the human bone morphogenic protein-7 complementary deoxyribonucleic acid into periosteal-derived rabbit mesenchymal stem cells. Bone morphogenic protein-7 secreting gene modified cells subsequently were expanded in monolayer culture, seeded onto polyglycolic acid grafts, implanted into a rabbit knee osteochondral defect model, and evaluated for bone and cartilage repair after 4, 8, and 12 weeks. The grafts containing bone morphogenic protein-7 gene modified cells consistently showed complete or near complete bone and articular cartilage regeneration at 8 and 12 weeks whereas the grafts from the control groups had poor repair as judged by macroscopic, histologic, and immunohistologic criteria. This is the first report of articular cartilage regeneration using a combined gene therapy and tissue engineering approach.

Gene-enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene.

Periosteum has cell populations, including osteoprogenitor and chondroprogenitor cells, that can be grown in cell culture and form both bone and cartilage under appropriate conditions. The authors have shown previously that cultured periosteal cells can be used in the tissue engineering of bone, and they demonstrated substantial bone formation in a rabbit cranial defect model. In the current study, principles of tissue engineering were combined with principles of gene therapy to produce cultured periosteal cells transduced retrovirally with the bone morphogenetic protein 7 (BMP-7) gene to be used in the treatment of bone defects. Human BMP-7 complementary deoxyribonucleic acid was generated from a cell line using reverse transcription polymerase chain reaction and cloned into a retroviral vector plasmid. Retroviral vector particles were then used to transduce New Zealand White rabbit periosteal cells. Transduced periosteal cells demonstrated substantial production of both BMP-7 messenger ribonucleic acid by Northern blot analysis and BMP-7 protein by enzyme-linked immunosorbent assay. These cells were then seeded into polyglycolic acid (PGA) matrices and used to repair critical-size rabbit cranial defects. At 12 weeks, defect sites repaired with BMP-7-transduced periosteal cells/PGA had significantly increased radiographic and histological evidence of bone repair compared with those defect sites repaired with negative control-transduced cells/PGA, nontransduced cells/PGA, PGA alone, or unrepaired defects. Thus, this study demonstrates successfully a tissue engineering approach to bone repair using genetically modified cells.

Biological Alchemy: Engineering Bone and Fat From Fat-Derived Stem Cells

Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. In this study the authors isolated these fat-derived stem cells successfully from Lewis rats and induced differentiation along adipogenic and osteogenic lineages in vitro and in vivo. Induction was stimulated by exposing stem cells to lineage-specific induction factors. Adipocyte-inducing media contained dexamethasone, insulin, and isobutyl-methylxanthine. Osteoblast inducing media contained dexamethasone, &bgr;-glycerophosphate, and ascorbic acid. Undifferentiated stem cells were maintained in minimal essential media alpha and fetal bovine serum. At 10 days, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil red O staining of lipid vacuoles. At 21 days, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix and immunohistochemical staining for osteocalcin. Differentiated cells were seeded at a density of 5 × 106 cells onto 15 × 15-mm polyglycolic acid grafts and implanted subcutaneously into three groups of Lewis rats: Group I contained undifferentiated stem cell grafts, group II contained adipocyte grafts, and group III contained osteoblast grafts. At weeks 4 and 8, in vivo fat formation was demonstrated in group II rats, as confirmed by Oil red O staining. At 8 weeks, group III rats demonstrated in vivo bone formation, as confirmed by the presence of osteocalcin on immunohistochemistry and the characteristic morphology of bone on hematoxylin–eosin staining. Group I rats demonstrated no in vivo bone or fat formation at either time interval. These results demonstrate the ability to isolate pluripotent stem cells from adipose tissue, to induce their differentiation into osteoblasts and adipocytes in vitro, and to form bone and fat subsequently in vivo. This is the first published report of in vivo bone formation from fat-derived stem cells. These cells may eventually serve as a readily available source of autologous stem cells for the engineering of bone and fat.

Repair of palatal bone defects using osteogenically differentiated fat-derived stem cells.

Background: Although autogenous bone grafting remains the standard in the reconstruction of bone defects, disadvantages may include limited amount of bone and donor-site morbidity. Tissue engineering approaches can potentially obviate these problems. Fat contains a population of stem cells that can be isolated and differentiated into various cell lines, including osteocytes, adipocytes, and myocytes, depending on the culture conditions. In this study, the authors used osteogenically differentiated fat-derived stem cells to repair rat palatal bone defects. Methods: Fat-derived stem cells were isolated, differentiated into osteocytes in osteogenic medium, and seeded onto poly-L-lactic acid scaffolds. Rat palatal bone defects were surgically made and animals divided into four groups according to the type of implant for bone repair: group I, empty defect; group II, poly-L-lactic acid without cells; group III, poly-L-lactic acid with undifferentiated fat-derived stem cells; and group IV, poly-L-lactic acid with osteogenically differentiated fat-derived stem cells. Palates were harvested at 6 or 12 weeks after implantation (n = 8 per group at each time interval). Hematoxylin and eosin staining, immunohistochemical staining for osteocalcin, and histomorphometric measurements of new bone were performed. Results: Defects in groups I, II, and III had no bone and were primarily filled with fibrous tissue. In contrast, there was substantial bone regeneration in group IV, which was statistically significant by histomorphometry compared with groups I, II, and III. Newly formed bone in group IV stained positive for osteocalcin. Conclusions: The authors successfully reconstructed palatal bone defects using absorbable three-dimensional scaffolds seeded with osteogenically differentiated fat-derived stem cells. This study demonstrates the feasibility of reconstructing bony defects with fat-derived stem cells.

The role of rigid skeletal fixation in bone-graft augmentation of the craniofacial skeleton

The type of fixation (rigid skeletal vs. wire) was assessed against embryologic origin (membranous vs. endochondral) and recipient site (depository vs. resorptive) as variables affecting inlay and onlay bone-graft survival in 20 mature dogs. Wet weight and volume measurements were made at operation and at sacrifice (16 weeks). The results were as follows: (1) Rigid skeletal fixation increased bone-graft volume survival over wire fixation (p less than 0.05). (2) Fixation (i.e., rigid skeletal) and embryologic origin (i.e., membranous) were equal determinants of bone-graft volume survival (p less than 0.001); the recipient site was not significant for onlay bone graft survival. (3) Embryologic origin was the only significant determinant of weight survival (p less than 0.001). (4) Inlay bone grafts demonstrated greater weight and volume survival than onlay bone grafts (p less than 0.05). (5) Histologic and microradiographic studies demonstrated bony union of bone grafts fixed with rigid skeletal fixation, while fibrous union predominated in bone grafts fixed with wire technique.

Gene-enhanced tissue engineering : Applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene

The treatment of difficult wounds remains a considerable clinical challenge. The goal of this study was to determine whether genetic augmentation of dermal cells on resorbable matrices can stimulate the healing process, leading to increased tissue repair in a rat full-thickness excisional wound repair model. The human platelet-derived growth factor B (PDGF-B) gene was the initial gene chosen to test this hypothesis. The human PDGF-B gene was obtained from human umbilical vein endothelial cells (HUVEC) by reverse transcriptase-polymerase chain reaction, cloned into retroviral vectors under control of either the cytomegalovirus promoter or the rat beta-actin promoter, and introduced into primary rat dermal cells. In vitro results demonstrate that rat dermal cells are transduced and selected readily using retroviral vectors, and engineered to secrete PDGF-B at a steady-state level of approximately 2 ng per milliliter culture per 1 million cells per 24 hours. Seeding of the gene-modified cells onto polyglycolic acid (PGA) scaffold matrices and introduction into the rat model resulted in substantially increased fibroblast hypercellularity over control wounds at both 7 and 14 days posttreatment. Our results demonstrate that gene augmentation of rat dermal fibroblasts with the PDGF-B gene introduced into this animal model via PGA matrices modulates wound healing and suggests that experimentation with additional genes for use separately or in combination with PDGF-B for additional, improved wound healing is warranted.

Tricalcium phosphate and osteogenin: A bioactive onlay bone graft substitute

The disadvantages of autogenous bone grafts has prompted a search for a dependable onlay bone graft substitute. A combination of tricalcium phosphate, a resorbable ceramic, and osteogenin, an osteoinductive protein, was evaluated as an onlay bone graft substitute in a rabbit calvarial model. Twenty-eight tricalcium phosphate implants (15 mm diameter x 5 mm; pore size, 100-200 microns) were divided into experimental and control groups and placed on the frontal bone of 14 adult New Zealand White rabbits. In the experimental animals, 185 micrograms of osteogenin was added to each implant. In the control animals, the implants were placed untreated. Implants were harvested at intervals of 1, 3, and 6 months, and evaluated using hematoxylin and eosin histology, microradiography, and histomorphometric scanning electron microscope backscatter image analysis. At 1 month there was minimal bone ingrowth and little tricalcium phosphate resorption in both the osteogenin-treated and control implants. At 3 months, both the osteogenin-treated and control implants showed a modest increase in bone ingrowth (8.85 percent versus 5.87 percent) and decrease in tricalcium phosphate (32.86 percent versus 37.08 percent). At 6 months, however, the osteogenin-treated implants showed a statistically significant increase in bone ingrowth (22.33 percent versus 6.96 percent; p = 0.000) and decrease in tricalcium phosphate (27.25 percent versus 37.80 percent; p = 0.004) compared with the control implants. The bone within the control implants was mostly woven at 6 months, whereas the osteogenin-treated implants contained predominantly mature lamellar bone with well-differentiated marrow. All implants maintained their original volume at each time interval studied. The tricalcium phosphate/osteogenin composite, having the advantage of maintaining its volume and being replaced by new bone as the tricalcium phosphate resorbs, may be applicable clinically as an onlay bone graft substitute.

Lentiviral transfection with the PDGF-B gene improves diabetic wound healing.

Background: The treatment of diabetic wounds remains a difficult challenge. The present study investigates whether platelet-derived growth factor (PDGF) lentiviral gene therapy can improve diabetic wound healing in the diabetic db/dbmouse. Methods: PDGF cDNA was cloned and lentiviral vectors were constructed with either the PDGF-B or green fluorescence protein (GFP) gene. A 2 × 2-cm full-thickness dermal wound was made on each db/db mouse. Animals were divided into three groups, with eight animals in each group as follows: group I, empty wound; group II, lentiviral PDGF; and group III, lentiviral GFP. Lentiviral vectors were injected into the wounds and healing was assessed at 21 days. Harvested wounds were assessed for residual epithelial gap, granulation tissue area, PDGF expression, collagen formation (picrosirius red), and angiogenesis (CD31 staining). Results: Lentiviral vectors were constructed and transfected dermal fibroblasts demonstrated in vitro production of PDGF mRNA as measured by reverse-transcriptase polymerase chain reaction. Immunohistochemistry for PDGF confirmed successful in vivo transfection of the PDGF gene. At 21 days, reepithelialization and granulation tissue area were similar in all groups. However, there was a statistically significant increase in angiogenesis and substantially thicker, more coherently aligned collagen fibers in the PDGF group compared with controls. Conclusions: PDGF lentiviral vectors were successfully transfected into the regenerated dermis in diabetic wounds. Although reepithelialization was similar among the groups, there was enhanced angiogenesis and collagen deposition in the lentiviral PDGF group. These results demonstrate that lentiviral PDGF transfection of the diabetic wound enhances PDGF production, improves vascularization and collagen organization, and has potential clinical applications in diabetic wound treatment.

The le fort III advancement osteotomy in the child under 7 years of age

This is a longitudinal study of 12 patients with craniofacial synostosis syndromes (Crouzons, Aperts, Pfeiffers) who underwent Le Fort III advancement under the age of 7 years (average age 5.1 years, range 4.0 to 6.7 years). The average follow-up was 5.0 years and included clinical, dental, and cephalometric examinations according to a prescribed protocol. The study demonstrated that the procedure could be safely performed in the younger child with an acceptable level of morbidity. There was a remarkable degree of postoperative stability of the maxillary segment. However, although vertical (inferior) growth or movement of the midfacial segment was demonstrated, there was minimal, if any, anterior or horizontal growth. Any occlusal disharmony developing during the period of follow-up could be attributed to anticipated mandibular development and could be corrected by orthognathic surgery. The roles of surgical overcorrection and anterior-pull headgear therapy after release of intermaxillary fixation are also discussed. The Le Fort III osteotomy is justifiably indicated during early childhood for psychological and physiologic reasons.