Eyal Ron

Biotechnology and bone graft substitutes

Trauma, disease, developmental deformities, and tumor resection frequently cause bone defects that seriously challenge the skills of orthopedic and maxillofacial surgeons. Currently, repairing osseous deficiencies involves various medical surgical techniques, including autogenous grafts, allografts, internal and external fixation devices, electrical stimulation, and alloplastic implants. The existing technology, though effective in many cases, still is beset with numerous difficulties and disadvantages. A critical need for improved treatment methods exists today. Biotechnology now provides access to new bone repair concepts via administration of protein growth and morphogenic factors. Implantable device and drug delivery system technologies also have advanced. The converging biopharmaceutical, device, and delivery technologies represent an opportunity to improve the quality of health care for individuals with orthopedic and maxillofacial deficiencies. This report reviews current concepts in fracture healing and bone repair and examines existing treatment modalities. It also addresses novel protein drugs that stimulate osseous regeneration and delivery systems for these drugs.

Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier

Recombinant human bone morphogenetic protein-2 was evaluated in maxillary alveolar clefts in 24 adult, skeletally mature Foxhound dogs. Bilateral clefts were prepared, 1 cm in bony width, lined with healthy epithelium with functional teeth on each side, and were expected not to heal spontaneously with new bone. Preparation of bilateral clefts in 24 dogs permitted 48 recipient sites divided evenly among four treatment and two time periods (2 and 4 months), yielding six replicates per treatment per time. The overall goal for the study was to regenerate bone in the cleft using one of three treatments: (2) 200 microgram recombinant human bone morphogenetic protein-2 combined with the copolymer poly(lactide-co-glycolide) and autogenous blood, (2) poly(lactide-co-glycolide) and autogenous blood, or (3) an autograft from the posterior iliac crest. A fourth group consisted of untreated alveolar cleft defects. At designated times, dogs were euthanized, and the recipient beds with contiguous bone were recovered, processed, and assessed radiographically and histologically. Autograft-treated defects had more bone than other treatments at 2 months; however, by 4 months, there were no differences among treatments, except for the poly(lactide-co-glycolide) group, which had the least amount of bone. Response to the recombinant human bone morphogenetic protein-2 may have been suboptimal either because the dose was too low or because the poly(lactide-co-glycolide)-autogenous blood delivery system did not temporally maintain and spatially position recombinant human bone morphogenetic protein-2 at the recipient bed. In addition, the development of a nonhealing, critical-sized defect in the maxilla of the dog appears to require a more aggressive resection of bone to preclude spontaneous osseous regeneration.

Recombinant human bone morphogenetic protein-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 µg/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-β1 (TGF-β1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-β1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 µg/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-β1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo. (Mol Cell Bioche m 167:31-39, 1997)

Poly(α-hydroxy acid) carrier for delivering recombinant human bone morphogenetic protein-2 for bone regeneration

Abstract Autografting is the most common treatment to correct osseous defects, with donor sites of calvarium, iliac crest, and rib used most often [1]. While varying degrees of ‘success’ have been reported with this therapy, a universal concern is donor site morbidity. Allogeneic bank bone is an alternative to the autograft and while donor site morbidity is not an issue, bank bone has liabilities that make it less effective than the autograft. Therefore, an off-the-shelf product to replace these therapies would improve the quality of care for patients. Towards the development of such a product, we report on two studies that assessed a combination of recombinant human bone morphogenetic protein-2 delivered to osseous wound sites with a poly(a-hydroxy acid) carrier: poly(lactide-co-glycolide). Results from these studies indicate bone repair can be accomplished with this combination. However, despite a rich therapeutic promise, clinical utility must be amplified. Therefore, the focus of our attention in the future will be the delivery system.

Osseous regeneration in preclinical models using bioabsorbable delivery technology for recombinant human bone morphogenetic protein 2 (rhBMP-2)

Abstract Novel delivery systems for the bone morphogenetic protein rhBMP-2, consisting of poly( d,l -lactide-co-glycolide) porous microspheres as bioabsorbable filling material with either autologous blood clot or carboxymethylcellulose as binding agent, were tested in several preclinical models including a rat calvarial defect model, a rabbit radius segmental defect model, and a rabbit ulna segmental defect model. In the rat calvarial defect model, these novel delivery systems were shown to be comparable to inactivated collagenous bone matrix as a matrix for rhBMP-2 delivery at the appropriate dose. The incidence of union in the rabbit long bone studies exhibited a rhBMP-2 dose-response, achieving an incidence of union of nearly 100% at the higher doses tested. Histological evaluation showed remodelling of newly generated bone, and biomechanical testing found that the healed limbs were as strong as untreated control limbs. These studies demonstrate that it is possible to obtain osseous regeneration of critical-size defects by combining rhBMP-2 with synthetic delivery systems.

Study of the Breakup Under Shear of a New Thermally Reversible Water-in-Oil-in-Water (W/O/W) Multiple Emulsion

AbstractPurpose. Thickening of the external aqueous phase of W/O/W multiple emulsions is essential to increase the release under shear. However, it leads to globules bursting during fabrication. To reduce this problem, we have tested a novel thermally reversible hydrogel, EMP hydrogel. This way, the corresponding multiple emulsion (EMPME) would gel only at skin temperature, which may increase the active ingredient delivery when topically applied. Methods. Samples were sheared at different shear rates and temperatures (20, 30, and 35°C) with a controlled rheometer. A granulometric analysis was then performed with a laser diffraction granulometer, to assess the break up as a function of the shear rate at the three temperatures. Conductometric measurements (CDM 230 conductometer) provided the corresponding release curves. Results. As we expected, EMPME exhibited a thermally reversible behavior. Compared to a reference emulsion thickened by carbopol, this new thermo–sensitive multiple emulsion displayed higher break up and fraction released at 35°C. Conclusion. The first thermally reversible multiple emulsion has been developed in the present work. This one presents interesting advantages: (1) an easy fabrication process with a higher entrapment yield and (2) a higher fraction released at 35°C compared with the reference emulsion.

A novel approach to administration of peptides in women: Systemic absorption of a GnRH agonist via transvaginal ring delivery system

UNLABELLED trans-Epithelial delivery of medication across the vagina has proven successful for administration of small, lipophilic molecules such as sex steroids. However, little information is available regarding the vaginal delivery of larger and more polar molecules that currently require parenteral administration because the vaginal epithelium is perceived as a barrier to absorption of larger molecular weight (MW) molecules. Six healthy women underwent administration of 18 or 36mg of leuprolide, a GnRH agonist and a larger MW peptide, via a novel ethylene vinyl acetate (EVA) ring transvaginal drug delivery system (TVDS). Serum levels rose within 8h following insertion: low dose at 310pg/ml and high dose at 1220pg/ml, i.e. levels typically following parenteral injections of leuprolide. GnRHa biological activity was validated by secretion of gonadotropins and sex steroids. These results demonstrate that the non-keratinized vaginal epithelium permits a rapid absorption of a biologically active peptide and that there is significant potential for a novel TVDS to deliver peptides and possibly other macromolecules therapeutically. SIGNIFICANCE STATEMENT Current routes of administration of medications can include oral, subcutaneous, intravenous, intramuscular, transcutaneous, etc. Many of these approaches have limitations, including pain, poor tolerability, lack of adherence, and inadequate delivery. Peptides, in particular, cannot typically be given orally because they are broken down in the intestinal tract before they are absorbed. While the skin is an attractive way to deliver medications, its superb intrinsic barrier function often makes this route untenable at times. The vaginal epithelium, in contrast, is not keratinized and can allow absorption of other molecules. In this study, we demonstrate that a novel transvaginal drug delivery system (TVDS) is capable of delivering peptide therapeutics to women in a non-parenteral fashion as demonstrated by both blood levels and biologic effects of its delivery.