Platelet-derived growth factor inhibits bone regeneration induced by osteogenin, a bone morphogenetic protein, in rat craniotomy defects.

Platelet-derived growth factor (PDGF) is a potent moderator of soft tissue repair through induction of the inflammatory phase of repair and subsequent enhanced collagen deposition. We examined the effect of recombinant BB homodimer PDGF (rPDGF-BB) applied to rat craniotomy defects, treated with and without bovine osteogenin (OG), to see if bone regeneration would be stimulated. Implants containing 0, 20, 60, or 200 micrograms rPDGF-BB, reconstituted with insoluble rat collagenous bone matrix containing 0, 30, or 150 micrograms OG, were placed into 8-mm craniotomies. After 11 d, 21 of the 144 rats presented subcutaneous masses superior to the defect sites. The masses, comprised of serosanguinous fluid encapsulated by fibrous connective tissue, were larger and occurred more frequently in rats treated with 200 micrograms rPDGF-BB, and were absent in rats not treated with rPDGF-BB. The masses underwent resorption within 28 d after surgery. OG (2-256 micrograms) caused a dose-dependent increase in radiopacity and a marked regeneration of calcified tissue in a dose-dependent fashion within defect sites. However, OG-induced bone regeneration was inhibited 17-53% in the presence of rPDGF-BB. These results suggest that rPDGF-BB inhibited OG-induced bone regeneration and stimulated a soft tissue repair wound phenotype and response.

Establishing an immortalized human osteoprecursor cell line: OPC1.

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.

Gene therapy approaches for modulating bone regeneration.

Following injury, bone has the ability to regenerate itself to a form and function nearly indistinguishable from the pre-injury state. However, if the injury is beyond a critical limit, recovery will not occur without therapeutic interventions. Autografts and implants with banked bone continue as the treatments of choice, although each exhibits limitations and liabilities. Alternatives have included the utilization of bone-graft substitutes that may incorporate bone derivatives and soluble signaling molecules such as mitogens and morphogens. In addition, an evolving treatment modality, gene therapy, offers an exciting avenue for bone regeneration. This review presents some of the current concepts for developing a rational gene therapy approach in bone regeneration.

Initial biocompatibility studies of a novel degradable polymeric bone substitute that hardens in situ.

Clinical management of osseous defects often requires bone grafts. The standard for treatment is autogenous bone harvested from sites such as either the iliac crest or the outer table of the calvaria. In addition to the problem of donor site morbidity and the limited supply of graft material, there is the additional operating time associated with harvesting procedures. A synthetic, bone graft substitute that can match the clinical performance of autogenous bone could alleviate these deficiencies. Therefore, a polymeric bone substitute was developed that consists of a four-armed star polymer of poly(dioxanone-co-glycolide) endcapped at each termini with a biocompatible lysine-based diisocyanate crosslinker. The polymer can be mixed with inorganic fillers such as either hydroxyapatite or tricalcium phosphate to form either injectable or moldable putty. The addition of a catalyst (for example, diethylaminoethanol and water) to the polymer produces a crosslinking reaction causing the combination to harden. This reaction is nontoxic, normo-thermic and can be performed in situ. During the course of the polymerization, carbon dioxide is liberated, producing an interconnected porous network within the implant, suitable for bone ingrowth. This paper will describe a preliminary biocompatibility assay of the bone substitute.

Therapeutic potential of bone morphogenetic proteins.

Recently, there has been substantial progress in the area of bone morphogenetic protein (BMP) research. This review serves as an up-to-date summary of the history of BMPs, the mechanisms of BMP signalling and the role of BMPs in adipose, kidney, liver, bone and nervous system. The potential of BMPs as therapeutic agents will also be discussed.

Poly(alpha-hydroxy acids): carriers for bone morphogenetic proteins.

A broad spectrum of cells and cell products is associated with bone homeostasis and the renewal of bone following injury. The coupled interactions among cells provide the power behind sculpting of bone, sustaining form, and ensuring functionality. Local and systemic regulatory molecules (e.g. growth factors, hormones) direct cellular interactions through autocrine, paracrine, and hormonal pathways. Recently, genes for a class of osteogenic regulatory molecules have been cloned, and gene product expression has enabled investigators to assess safety and efficacy in animal studies. The molecules are known as bone morphogenetic proteins (BMPs). Therapeutic applications of BMPs depend on a carrier system. A carrier could spatially and temporally localize BMP for regional needs and be custom-tailored for acute craniofacial applications or for recalcitrant extremity non-unions. The poly(alpha-hydroxy acids) (PHAs) may be suitable for these applications. Therefore, the purposes of this paper are (i) to mention, briefly, basic concepts of the bone wound continuum and the possible therapeutic roles of BMPs; (ii) to outline several properties of selected PHAs relevant to bone regeneration dynamics; and (iii) to review selected preclinical studies with PHAs.

An osteogenic cell culture system to evaluate the cytocompatibility of Osteoset, a calcium sulfate bone void filler.

The purpose of the study was to describe a convenient, reliable and quantitative in vitro assay system to assess the cytocompatibility of a calcium sulfate bone filler on two osteogenic cell lines and primary osteoblasts. The hypothesis was that the bone void filler, OsteoSet pellets, would not impact adversely on cell proliferation kinetics or osteogenic potential of selected cells. The hypothesis was tested by standard in vitro methodology of placing OsteoSet pellets either directly in contact with osteogenic cells, or by compartmentalizing within transwell - clear microporous membrane inserts. Data analyses were accomplished with appropriate post hoc statistics (p < or = 0.05). In the presence of the OsteoSet pellets, the cell lines exhibited a decrease in cell proliferation at days 4 and 7, independent of either cell type or tissue culture medium. A decrease in the alkaline phosphatase enzyme activity occurred in the osteogenic cell lines maintained for 9 and 16 days in the presence of the OsteoSet pellets. However, with the exception of the MC3T3E-1 line, no differences were observed with respect to calcium deposition (mineralization) by day 16. Intact human osteocalcin release data for the human-derived OPC1 line and the primary osteoblasts was inconclusive as the OsteoSet pellets may interact with the osteocalcin secreted into the tissue culture medium. The present studies describe a cell culture system to assess the cytocompatibility of bone-graft substitutes with osteogenic cells by compartmentalizing material from direct cell contact (in transwells), and additionally, by evaluating direct cell/biomaterial interactions.

Options for tissue engineering to address challenges of the aging skeleton.

There will be more than 52 million Americans over the age of 65 by the year 2020 (U.S. Census Bureau). Regenerating form and function to bone defects in an elderly, osteoporotic population of this magnitude will be a daunting challenge. Tissue engineering options must be considered to answer this challenge. Options can include gene transfer technology, stem cell therapy, and recombinant signaling molecules. An additional component will be a carrier that localizes, protects, predictably releases cues and cells, as well as establishes an environment for restoring osseous form and function. The purposes of this article are to present an overview of the bone regenerating decrement affecting osteoporotic, elderly patients and to highlight some tissue engineering options that could offset this decrement.

Tissue engineering of bone in the craniofacial complex.

Tissue engineered therapies to regenerate bone in the craniofacial complex will probably include combinations of BMP-like molecules, a BMP-responsive set of cells (both endogenous and exogenous), and packaging in a surgically convenient format. In this report we have described our work with OPCs, BMP, and polymer: components suitable for tissue engineering.

Macrophysiologic roles of a delivery system for vulnerary factors needed for bone regeneration.

Traditional histology identifies three components of bone: cells, an extracellular mineralized organic matrix, and a lymphatic-vascular component. Specialized bone cells known as osteoblasts promote bone regeneration. Clinically, this property has been exploited by surgeons with autografts and bank bone preparations to restore deficient form and function to almost every aspect of the skeleton. Unfortunately, these therapies can be inadequate for patients with panskeletal trauma. Therefore, a suitable alternative may be a laboratory-derived product consisting of a vulnerary factor and delivery system. The integration of a laboratory-engineered product in an osseous wound environment is a formidable challenge demanding a keen appreciation of the product's macrophysiologic roles in wound healing biology. Consequently, the purposes for this paper are 1) to define briefly macrophysiology relevant to a delivery system for vulnerary molecules and bone regeneration; 2) to review a key family of bone regenerating molecules, the bone morphogenetic proteins (BMPs); and 3) to relate delivery system engineering with bone regeneration.

Osseous wound healing with xenogeneic bone implants with a biodegradable carrier.

Human antigen-extracted, autolyzed (AA) bone and a bovine bone morphogenetic protein were prepared as implants within biodegradable carriers and compared with autogenous bone grafts and controls in the healing of critical-size bony defects in nonhuman primates. The treated craniotomy sites were studied 3 and 6 months after surgery; radiodensity and volume of newly deposited trabecular bone were assessed by radiomorphometric and histomorphometric methods, respectively. There was no evidence of adverse immunologic response to the experimental implants. The autografts resulted in the greatest volume of new bone formation (p less than 0.01), but the AA implants elicited a significantly greater response than either the bovine bone morphogenetic protein derivatives or the controls (p less than 0.05). By 6 months, the AA derivatives had healed with actively coalescing islands of new bone, displaying normal-appearing outer and inner tables along with well-developed marrow cavities. It appears that xenogeneic AA implants have the ability to elicit an excellent osseous response in critical-size calvarial wounds. In addition, the carrier polymer for the implants acted as an effective soft-tissue spacer before being absorbed.

Incorporation of polylactide-polyglycolide in a cortical defect: neoangiogenesis and blood supply in a bone chamber.

Erodible polymers are an alternative to metals for fracture fixation (for example, in the malleolus) and for maxillofacial reconstruction. In this study, the vascular response to eroding polylactide-polyglycolide copolymer threads was observed chronically in a bone chamber implant, with use of intravital microscopy. A bone chamber implant loaded with 100 microns thick polylactide-polyglycolide threads was implanted into the right tibia in 15 mature female New Zealand White rabbits. Periodic intravital microscopic observations were performed from the third to the tenth or twelfth week after implantation. Vascularization, blood flow, and trabecular growth into the chambers from the medial cortex were recorded on videotape and analyzed using digital image processing. A statistically significant delay of neo-osteogenesis in the presence of this copolymer was described in an earlier report. The present report describes the measures of neoangiogenesis and blood supply; there was a significant delay in neoangiogenesis. It is suggested that both delayed angiogenesis and osteogenesis were secondary consequences of the macrophage response to slowly eroding poly-L-lactide crystal nanoparticles and the influence of reduced nutrient exchange. The lesser effect on blood supply and vascular volume fraction was seen to be linked to the slowing down of angiogenesis, as the latter allowed vessels to mature, with a widening of their calibers. This homeostatic adjustment was interpreted as being only partially successful in restoring control levels of oxygen delivery, because resulting increases in vessel surface area did not reach control levels. Thus, in the presence of eroding polylactide-polyglycolide, the oxygen supply and extravasation of other nutrients may be below normal during healing phases when the need is critical.

Bone morphogenetic proteins: an update on basic biology and clinical relevance.

The regeneration of bone is a remarkable, complex physiological process, and BMPs are a formidable clinical tool to promote its regeneration. By defining roles played by BMPs in developmental biology and bone regeneration, significant progress has been made to identify cell-signaling molecules and their regulators. For example, the regulators of BMPs that include noggin, chordin, cerberus, dan, and gremlin may be harnessed as therapies to offset calcification encountered after total hip arthroplasties. Furthermore, exploiting BMPs and Smads may generate new therapeutic options for bone repair. Another compelling clinical consideration is the trans-acting factor osteoblast-specific factor-2, which can promote osteoblast differentiation. Moreover, the affiliation of osteoblast-specific factor-2 with heritable disorders merits exploration. A recognized daunting challenge includes a carrier/delivery system for the powerful morphogenetic therapeutic tools, as well as osteoprogenitor cells and intracellular transduction and transcriptional factors. In addition, the long-term effects of administering superphysiological doses of rhBMPs to patients must be assessed systematically. A new generation carrier/delivery system may be the answer to offset dosing liabilities as well as to provide residence for exogenous, BMP-receptive osteoprogenitor cells (111,112). The areas highlighted in this review offer fertile territory for thought and research to develop rational clinical treatments to promote bone regeneration and to understand some of the biological roles of BMPs.

Rabbit calvarial wound healing by means of seeded Caprotite scaffolds.

Autologous bone is the most successful bone-grafting material; however, limited supply and donor site morbidity are problematic. Synthetic bone substitutes are effective, but healing is slow and unpredictable. Osseous wound healing may be enhanced if bone substitutes are combined with autologous bone marrow cells. To test this hypothesis, we created 40 calvarial defects in 20 12-week-old New Zealand White rabbits, divided into four groups: (1) unrepaired controls, (2) autologous bone grafts, (3) unseeded Caprotite (a polymer-ceramic composite) grafts, and (4) Caprotite grafts seeded with autologous bone marrow stromal cells. CT scans were obtained at 0, 6, and 12 weeks post-operatively, and defects were harvested for histology. Defects repaired with autologous bone had significantly (p < 0.05) more bone than the other three groups, although seeded Caprotite defects showed different wound-healing sequelae. Results suggest that seeded Caprotite scaffolds did not significantly enhance osseous defect healing compared with controls.

Bone formation with use of rhBMP-2 (recombinant human bone morphogenetic protein-2).

We examined the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2), delivered in a porous poly(DL-lactic acid) implant, on bone formation in a critical-sized defect in the radial diaphysis in rabbits. A unilateral segmental defect, twenty millimeters long, was created in the radius in ninety-six skeletally mature New Zealand White rabbits. Forty-eight rabbits were evaluated at four weeks and forty-eight, at eight weeks. Six groups were studied at each time-period. The defect was left empty in one group (control), the defect was filled with an autogenous corticocancellous bone graft in one group, and the defect was filled with a porous poly(DL-lactic acid) implant containing zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 (one group each). Radiographs of the defects were made every two weeks. The percentage of the total area of the defect that was radiopaque was determined with use of computerized radiomorphometry, and this percentage was used as a quantitative measure of the extent of new-bone formation in the defect. There were time and dose-dependent responses to rhBMP-2 for as long as four weeks; thereafter, the effects of seventeen, thirty-five, and seventy micrograms of rhBMP-2 were independent of dose and time (p < or = 0.05). The defects that had been treated with either thirty-five or seventy micrograms of rhBMP-2 had a significantly greater (p < or = 0.05) area of radiopacity than the defects that had been treated with either zero or seventeen micrograms of rhBMP-2. No significant difference could be found between the defects treated with thirty-five or seventy micrograms of rhBMP-2 and the defects filled with an autogenous graft. Healing and bone formation were examined histologically and histomorphometrically as well. At four weeks, polarized light microscopy revealed remnants of poly(DL-lactic acid) only in the defects that had been filled with an implant containing zero micrograms of rhBMP-2. At eight weeks, regardless of the dose of rhBMP-2, poly(DL-lactic acid) was not visible on histological examination. The presence of multinucleated giant cells was the hallmark of the inflammatory response elicited by poly(DL-lactic acid). At four and eight weeks, macrophages and lymphocytes were also present. The intensity of the cellular response at four weeks suggested an inverse relationship between these cells and the dose of rhBMP-2 -- that is, there appeared to be more multinucleated giant cells in defects treated with zero micrograms of rhBMP-2 than in defects treated with seventy micrograms of rhBMP-2. At eight weeks, multinucleated giant cells were rare in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2. Histomorphometric data at four and eight weeks indicated that the amount of bone formation in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2 was equivalent to the amount in the defects treated with an autogenous graft and was significantly less (p < or = 0.05) in the untreated defects and the defects treated with zero micrograms of rhBMP-2 (p < or = 0.05). By eight weeks, only thirty-five and seventy micrograms of rhBMP-2 had restored cortices and marrow elements.

Controlled release from coated polymer microparticles embedded in tissue-engineered scaffolds.

The controlled release of proteins in tissue-engineered implants is being examined with the potential application to improve vascularization and hasten tissue growth. Bovine serum albumin (BSA), was encapsulated within poly(D,L-lactic-co-glycolic acid) [PLGA] microparticles. The microparticles were coated with poly(vinyl alcohol) and incorporated into PLGA tissue-engineered scaffolds during fabrication. The release of BSA from PLGA microparticles, coated PLGA microparticles, and microparticles embedded in a porous PLGA scaffold was measured. We have developed a novel approach that will permit incorporation of coated polymeric microparticles during PLGA scaffold fabrication. Growth factors or drugs could be incorporated into the microparticles resulting in a long-term, controlled release.

Cranial, iliac, and demineralized freeze-dried bone grafts of the mandible in dogs.

Both autogenous bone grafts and demineralized freeze-dried allogeneic bone implants were evaluated for mandibular reconstruction. Four-centimeter segmental defects of the midbody of the edentulous mandible were reconstructed in 36 dogs, with specimens recovered at 3 and 6 months and quantitatively compared for total and new bone by histomorphometric analysis. Autogenous grafts consisted of corticocancellous cranial block (CB), corticocancellous iliac block (IB), and particulate cancellous iliac marrow (PM). The allogeneic bone was demineralized and freeze-dried, and consisted of particulate cortical endochondral bone (FP), cranial cortical block (FCB), and iliac cortical block (FIB). Clinically and histomorphometrically, results appeared to indicate that (1) CB compared favorably with IB at 3 and 6 months for total bone, but IB showed a trend for more new bone formation at 6 months, a trend that may be due to the thicker cortical component of CB, which requires longer time periods to remodel than the cancellous rich IB; (2) FP failed to achieve bony union at 3 months, with inadequate rates of new bone formation; and (3) FCB and FIB compared favorably for total bone with CB and IB at 6 months, although new bone for autogenous CB and IB was 26.9% and 45.4%, while new bone for allogeneic FCB and FIB represented only 7.9% and 17.4%.

Wound healing. Relationship of wound closing tension to scar width in rats.

A study was conducted to better define the relationship between closing tension and the resulting scar width of incisional wounds. Five groups of 10 hairless rats each were studied. Transverse wounds were created and closed on the back of each rat, with closing tension varied by excising amounts of skin in widths of 0 (control), 15, 30, 45, and 60 mm. At 28 days, the scar width was measured by three methods: digital caliper, photographically, and histologically. Results showed that wounds closed under the highest tension (60-mm excision group) had significantly wider scars than controls by all three measurement techniques. Regression analysis of the caliper scar width as determined by squaring the closing tension resulted in a nonlinear equation resembling an exponential curve that "best fit" the variables.

The effects of ethylene oxide sterilization on the in vitro cytotoxicity of a bone replacement material.

The effect of degassing on the cytotoxicity of an ethylene oxide (EtO)-sterilized copolymer constituent of a bone replacement material, was determined using the (51)Cr release assay. An initial experiment used mouse L929 cells and a copolymer with an ambient pressure and temperature (APT) degassing time of 24 hr. Both copolymer and nylon control discs caused a significantly (P

Comparison of porous bone mineral and biologically active glass in critical-sized defects.

Several materials have been proposed as therapies to augment alveolar bone and to promote periodontal regeneration. However, there are an insufficient number of studies that effectively evaluated these therapies. Consequently, the purpose of this study was to compare bone regeneration promoted by porous bone mineral and biologically active glass. Unilateral critical-sized defects (CSDs) were prepared in the radii of 24 rabbits, divided evenly between 2 time periods (4 and 8 weeks) and between 2 treatment groups (porous bone mineral and biologically active glass). Evaluations consisted of clinical examinations, standardized radiography at baseline and every 2 weeks thereafter, as well as histology and histomorphometry. Data were analyzed by an unpaired Student t-test with significance established at P < or = 0.05. We determined that CSDs treated with porous bone mineral were significantly more radiopaque than biologically active glass-treated sites at both 4 and 8 weeks. Moreover, the amount of new bone was significantly greater at both 4 and 8 weeks in the porous bone mineral groups than in the biologically active glass groups. We concluded that in the rabbit radius CSD wound model, porous bone mineral appears to be more effective than biologically active glass in regenerating bone.