Single-dose growth factor treatments to enhance cell recruitment and neotissue integration in an augmented microfracture cartilage repair model

Abstract

Focal cartilage defects caused by joint injury have a limited capacity to self-repair, and if left untreated, can lead to the early onset of osteoarthritis. The current gold standard of care, microfracture surgery, induces an endogenous repair response, but typically results in poorly integrated fibrocartilage, rather than native hyaline cartilage. The objective of this thesis was to test the hypothesis that a self-assembling peptide hydrogel functionalized with chemotactic and pro-anabolic growth factors and placed into the defect during surgery could induce migration of endogenous progenitor cells into the repair tissue. Since these progenitors are naturally accessed during microfracture surgery, clinical translation of this approach could ultimately steer repair to a more hyaline-like response. Poor cartilage repair is believed to be the result of an insufficient number of progenitor cells at the defect site. We hypothesized that the addition of a single dose combination of chemotactic growth factors (such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-P 1 (TGF-[beta]1), and heparin-binding IGF-l (HB-IGF-1)) premixed into a hydrogel scaffold could stimulate bone-marrow progenitor cell migration into the hydrogel. A novel 3D gel-to-gel migration assay, using (KLDL)₃ self-assembling peptide gels, demonstrated that the combination of PDGF-BB and TGF-[beta]1 induced significant migration into the gel compared to growth-factor free controls. Importantly, these growth factors were retained in the hydrogel and exhibited a slow release over 1-2 weeks. We also hypothesized that a brief enzymatic pre-treatment of the defect site could release proteoglycans from the walls of the surrounding native cartilage in a controlled manner, and thereby create space for newly synthesized repair tissue to anchor and integrate with this adjacent host cartilage. We used an in vitro model in which a cylindrical annulus of native cartilage was pre-treated with trypsin over a 2-minute period and then filled with a chondrocyte-seeded (KLDL) ³ hydrogel ftnctionalized with pro-anabolic HB-IGF-I that had been premixed into the gel. (This procedure was deemed to be clinically tractable by collaborating equine surgeons now using this approach in parallel animal studies.) Trypsin pre-treatment depleted proteoglycan content of adjacent cartilage in a controlled manner, and HB-IGF-l was found to be delivered to the surrounding cartilage from the peptide gel. HB-IGF-I was found to stimulate matrix biosynthesis both in the surrounding cartilage and the chondrocyte-seeded KLD scaffold, and to enhance mechanical integration. We further explored the uptake and diffusive transport properties of HB-IGF-l into cartilage motivated by the need to understand the pharmacokinetics of delivery from the repair construct to surrounding cartilage. The positively charged heparin-binding domain of HB-IGF-l results in high uptake into cartilage, making it an effective method of delivering the pro-anabolic attributes of IGF-1, which in its native form would be rapidly cleared from the joint. The observed high and rapid uptake of HB-IGF-l into cartilage will enable characterization of dosing for HB-IGF-l delivery to cartilage by either intra-articular injection or from implanted hydrogel scaffolds. In summary our results show that of (KLDL)₃ peptide hydrogel scaffolds can foster growth-factor induced progenitor cell migration to increase progenitor cell recruitment into a cartilage defect. Thus, the use of a peptide gel premixed with PDGF-BB and HB-IGF-l to enhance progenitor migration into the scaffold, combined with a trypsin pre-treatment to help promote subsequent integration, is a promising strategy towards improving integrative repair. The combination of these approaches is currently being tested in an in vivo rabbit model.