Apatite-polymer composites for the controlled delivery of bone morphogenetic proteins

Abstract

Current treatment of bone defects due to trauma, cancer, or degenerative spine diseases involves the implantation of a bone graft. Autografts, which are harvested from the patient's own body, are associated with problems of limited availability and surgical morbidity. The use of allografts obtained from donors is also not desirable due to the risks of disease transmission and the costs of maintaining bone banks. The ideal solution would be to regenerate native bone to fill the defects. A group of potent growth factors known as bone morphogenetic proteins (BMPs) have been hailed as alternatives to bone grafts due to their ability to elicit new bone formation. Clinical use of BMPs involves loading the protein solution onto collagen sponges and subsequent implantation. However, these conventional collagen carriers show rapid clearance of BMPs within [approx.] 2 weeks, whereas bone healing is a longer process, especially in higher mammals. The poor BMP retention in collagen sponges may explain the greater response variability in higher mammals, ranging from full bone bridging within weeks to no bone union. These sponges are also not capable of tunable or multifactor release that could benefit healing in certain anatomic sites, e.g. avascular sites and prolonged non-unions. Hence, the motivation of this thesis is to develop new carriers that allow more efficacious and flexible delivery of BMPs to achieve bone healing.

(cont.) The carrier should ideally exhibit (i) sustained release to maintain the response and activity of bone-forming cells, (ii) low initial burst to avoid adverse effects of a bolus administration and to conserve the expensive growth factor, and (iii) tunable release to meter out BMPs at the desired rate. In particular, tunable release and low burst release have long been challenges in controlled delivery systems. A carrier that can offer such temporal control will be highly valuable to the delivery of other therapeutic proteins, drugs and genes as well. To this end, we have devised a novel composite of two biomaterials with proven track records: poly(lactic-acid-co-glycolic acid) (PLGA) and apatite. The controlled release strategy was based on the use of a biodegradable polymer with acidic degradation products to manipulate the dissolution of the basic apatitic component. Proteins were pre-adsorbed onto the apatitic component such that as the apatite dissolved, proteins were released. Apatite-PLGA composites were formed into microparticles by a solid-in-oil-in-water emulsion process. In contrast to polymeric microparticles prepared by the conventional water-in-oil-in-water emulsion process, these composite microparticles exhibited zero-order, low burst release.

(cont.) Low burst release was attributed to the affinity of the apatite for the protein; until the apatite was dissolved, the protein was sequestered and prevented from premature release. Accordingly, the use of apatite singly as a carrier would have led to extremely slow release. A model protein, bovine serum albumin (BSA), and a therapeutic protein, recombinant human BMP-2 (rhBMP-2), were encapsulated in these apatite-PLGA composite particles. The release profile was modified systematically by changing variables that affected polymer degradation and apatite dissolution, such as polymer molecular weight, polymer hydrophobicity, apatite loading, and apatite particle size. An increase in polymer molecular weight, apatite loading or apatite particle size reduced the release rate of both BSA and rhBMP-2. Interestingly, increasing polymer hydrophobicity diminished the release of BSA, but enhanced the release of rhBMP-2. Slower polymer degradation associated with greater polymer hydrophobicity might have decreased the total amount of protein released, but preserved a larger bioactive fraction due to milder pH conditions within the particles. A suitable particle formulation for sustained rhBMP-2 delivery was identified as protein-sCAP-59 kD PLGA.

(cont.) When rhBMP-2 was encapsulated in these composite microparticles, it was released in a sustained fashion over 100 days. More importantly, the bioactivity of the protein was retained, as evaluated by its ability to induce the differentiation of mesenchymal stem cells toward the osteoblast lineage. Specifically, the levels of osteoblastic phenotype markers such as alkaline phosphatase (ALP) and osteocalcin were found to be significantly elevated compared to the controls. In contrast, rhBMP-2 released from conventional collagen sponges after 2 weeks did not increase the ALP expression over the controls. Protein-loaded composite microparticles were dispersed in secondary matrices, either gelatin or collagen sponges, for bone tissue engineering. Multifactor release from these scaffolds was possible through the incorporation of different sets of composite microparticles containing different proteins and exhibiting distinct release profiles. Collagen sponges injected with rhBMP-2-loaded composite microparticles were implanted in subcutaneous sites in rats. These composite collagen sponges stimulated a much higher degree of cellularity and vascularity than the controls without BMPs. The increased vascularity might be evidence of the angiogenic activity of rhBMP-2 at low concentrations in vivo.