Jeffrey O. Hollinger

Poly(α-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.

Carrier systems for bone morphogenetic proteins.

Bone deficits can regenerate inherently, although when the amount of bone loss exceeds a critical limit, pseudarthrosis and fibrosis occur. Therapeutic intervention either with an autograft or allogeneic bank bone are traditional options to promote regeneration to overcome critical limits. However, liabilities with traditional treatments have inspired investigators to develop alternatives, such as combinations of biomimetic scaffolds and osteogenic regulatory molecules. The class of osteogenic regulatory molecules known as the bone morphogenetic proteins has several members that stimulate bone regeneration. Therapeutic applications of bone morphogenetic proteins require a well characterized carrier system to ensure safe and effective presentation at the implant site. Several carrier systems have been used to evaluate the sustained release and implant retention of recombinant human bone morphogenetic protein-2. The carrier systems used in this study include type I collagen, poly(D,L-lactide), and deorganified bovine bone. Pharmacokinetics of recombinant human bone morphogenetic protein-2 released from these systems were characterized in the rat ectopic assay. Pharmacokinetics were influenced by the implant carrier. For example, sustained release occurred with the collagen sponge. The recombinant human bone morphogenetic protein-2 from deorganified bovine bone resulted in a burst release at the first collection interval, but thereafter, appeared to bind irreversibly to the morphogen. The poly (D,L-lactide) systems showed a dose dependent sustained release pattern. These results indicate the physicochemical characteristics of a carrier system for recombinant human bone morphogenetic protein-2 impact the release kinetics and may have a profound influence on clinical outcome.

Recombinant human bone morphogenetic protein‐2 and collagen for bone regeneration

The study reported describes a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen (C) to regenerate bone. Unilateral critical-sized defects (CSDs) were prepared in radii of 32 skeletally mature New Zealand white rabbits. Rabbits were divided evenly among four treatments: autograft, absorbable C (Helistat), 35 microg of rhBMP-2 combined with absorbable C (rhBMP-2/C), and untreated CSDs. The two euthanasia periods were 4 and 8 weeks. Radiographs were taken the day of surgery, every 2 weeks, and at term and the percent of radiopacity was measured. Data analysis revealed a time-dependent increase in the percent radiopacity with rhBMP-2/C. Histological examination revealed the rhBMP-2/C treatment regenerated osseous contour by 8 weeks. According to quantitative histomorphometry, the CSD and C groups had significantly less new bone than either autograft or rhBMP-2/C (p < or = 0.05). The results suggest that rhBMP-2/C could be an effective therapy to restore segmental bone defects.

Sustained release emphasizing recombinant human bone morphogenetic protein-2

Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems.

Calvarial Bone Repair with Porous D,L-Polylactide

Bone is a storehouse of biologic factors enabling it to regenerate without scar formation. Recombinant technology has made many of these factors available in significant quantity for therapeutic applications. However, a system to deliver recombinant bone-regenerating factors is needed. Biodegradable, biocompatible polymers have shown promise for delivering bone regenerative factors, such as bone morphogenetic protein. The polymer we selected to investigate was racemic D,L-polylactide. Our immediate objective was to engineer porous D,L-polylactide to promote bone ingrowth (osteoconduction). We tested the hypothesis that porous D,L-polylactide implanted in a standard intraosseous calvarial wound would not hinder but would support bone regeneration. Therefore porous polylactide disks (65% void volume) were manufactured with pores < or = 100 microns, < or = 200 microns, and < or = 350 microns; implanted in rabbits calvariae, and retrieved 1, 2, 4, and 6 months after insertion. Quantitative histomorphometry revealed a possible relationship in the amount of bone ingrowth with increasing pore size over time. The D,L-polylactide disks < or = 350 microns had the greatest quantity of bone ingrowth (< or = 0.05). However, a disturbing finding was the multinucleated giant cell response associated with all implanted disks. We speculate these cells may have produced an inhospitable environment stifling osteoconduction. Consequently, postsynthesis engineering refinements of D,L-polylactide to eliminate the giant cell response are crucial before loading with bone morphogenetic protein.

Impact of nicotine on bone healing.

A limited number of experimental animal studies and in vitro data confirm that nicotine impairs bone healing, diminishes osteoblast function, causes autogenous bone graft morbidity, and decreases graft biomechanical properties. Therefore, our long-term goal is to develop an effective therapy to reverse the adverse impact of nicotine from tobacco products. However, before accomplishing this goal, we had to develop an animal model. Our hypotheses were nicotine administration preceding and following autogenous bone grafting adversely affected autograft incorporation and depressed donor site healing in a characterized animal wound model. Hypothesis testing was accomplished in bilateral, 4-mm diameter parietal bone defects prepared in 60 Long-Evans rats (male, 35-day-old). A 4-mm diameter disk of donor bone was removed from the left parietal bone and placed in the contralateral defect. The donor site served as a spontaneously healing bone wound. The rats were partitioned equally among three doses of nicotine administered orally in the drinking water (12.5, 25, and 50 mg/L). For each dose, the duration and sequence of nicotine treatment followed four courses, including no nicotine and designated combinations of nicotine administration and abatement prior to and following osseous surgery. Experimental sites were recovered on 14 and 28 days postsurgery, responses quantitated, and data analyzed by analysis of variance and post hoc statistics (p < or = 0.05). We developed a convenient and effective osseous model, and the results validated our hypothesis that nicotine negatively impacts on bone healing.

The integrated processes of hard tissue regeneration with special emphasis on fracture healing

When bone is fractured, a sequence of dynamic events ensue to restore form and therefore function. Many key biologic cell regulators for these events have been identified, expressed through recombinant technology, and their roles posited. Moreover, the availability of recombinantly engineered molecules, such as the bone morphogenetic proteins with their potential to benefit patient care, has ushered in an important era in clinical dentistry that may eliminate either autografting or bank bone allografts. Therefore, in this review article, we have highlighted some of the exciting biologic regulators relevant to bone fracture healing and outlined the dynamic elements in this process.

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.