Organic-Inorganic Macroporous Cryogel Carrier for Bone Tissue Engineering Deepak Bushan Raina1, 2, Riya Dhar2, Lars Lidgren1, Hanna Isaksson1, Magnus Tägil1, Ashok Kumar2, *

1Lund University, Department of Orthopedics, Clinical Sciences Lund, Lund 221 85, Sweden, 2Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, India, * Corresponding author

Author Disclosures: LL is a board member of Bonesupport AB, Lund Sweden, Others: None Introduction Critical bone defects require medical intervention and invasive procedures. Tissue engineering aims to regenerate these defects by using bone-mimicking materials that can be of polymeric or composite nature. In this study, we aimed at producing a porous polymer composite bone substitute with osteogenic properties that can be used in non-unions or open fractures prone to non-healing. We describe the physico-chemical and carrier properties of composite polymeric cryogels made by a combination of organic and inorganic components. The cryogels consist of silk, chitosan and agarose as an organic backbone intended to provide mechanical strength, antibacterial and cell adhesive properties and lastly elasticity, respectively1. The inorganic components comprise either bioactive glass and hydroxyapatite (HA), or hydroxyapatite alone. The components were mixed to provide a milieu similar to native bone and enhance osteogenic differentiation of progenitor cells. Apatite has affinity for bisphosphonates2 and we aimed to investigate if these scaffolds can act as efficient carriers for co-delivery of recombinant human bone morphogenic protein-2 (rhBMP-2) and zoledronic acid (ZA). Methods Fabrication of cryogel scaffolds: Two types of scaffolds containing tetraethylorthosilicte (TEOS)-HA (THA) and only HA were fabricated by dissolving chitosan, silk and agarose in de-ionized water and 1% acetic acid. Bioactive glass was prepared by mixing TEOS and de-ionized water in equal ratios followed by addition of nitric acid as a catalyst and calcium chloride. HA was prepared by direct precipitation reaction of calcium hydroxide and ortho-phosphoric acid. All components were mixed together in THA scaffolds and all components except TEOS were mixed to make HA scaffolds. The constituents were mixed with 0.02% glutaraldehyde as a cross linker. The solution was then poured in plastic syringes and incubated at -20 °C for 12 h. The gels were later thawed and freeze dried for further analysis. Physico-chemical characterization: The physico-chemical properties of the scaffolds were analyzed using Fourier transform infrared (FTIR) spectroscopy to analyze the chemical structure of the crosslinked gels, thermo gravimetric analysis (TGA) for thermal stability of the materials, degradation analysis and also by performing energy dispersive X-ray (EDX) spectroscopic analysis for confirming the presence of inorganic constituents like bioactive glass and HA. Cell-material interaction: The biocompatibility of the materials was analyzed by accessing the proliferation of mesenchymal stem cells (MSCs) from rats and C2C12 mouse myoblasts using MTT assay. Adhesion of cells on the material was assessed using scanning electron microscopy (SEM) and DAPI. Moreover, due to the presence of bioactive components like bio-glass and HA, we also studied the alkaline phosphatase activity of MSCs and C2C12 cells seeded on the biomaterials as a cue to osteogenic differentiation. In-vivo delivery of rhBMP-2 and ZA: In order to assess the carrier properties of the biomaterial, we chose THA scaffolds for delivery of rhBMP-2 (10 µg) and ZA (10 µg) in a muscle pouch model in sprague dawley rats3.   The bone formation on the scaffolds was analyzed using micro-CT, histology, SEM and EDX. Three groups (n=6 in each) including scaffold alone, scaffold + rhBMP-2 and scaffold + rhBMP-2+ZA were used. The study was approved by the local ethics committee (M124-14). Results Physico chemical characterization: FTIR results clearly showed the crosslinking of polymeric components with a major vibration at 1630-1680 cm-1 indicating amide linkage. The vibrations at 800 cm-1 and 1000-1300 cm-1 show the presence of bioactive glass. Peaks between 900-1200 cm-1 confirm the presence of phosphate components while 1350-1550 cm-1 shows carbonate vibrations confirming the presence of both bio-glass and HA. Both THA and HA scaffolds were temperature stable and indicated minimal loss (<3%) at 37 °C. A sharp reduction of nearly 15% in original weight was seen around 100 °C. The scaffolds gradually resorb over time with nearly 60% of the scaffold resorbed at 84 days. EDX analysis re-confirmed the presence of bioactive glass and HA (peaks for Si, Ca and P) in THA scaffolds and HA (peaks for Ca and P) in HA scaffolds. Cell-material interactions: Both MSCs and C2C12 cells were compatible with THA and HA scaffold. C2C12 cells proliferated faster on plastic plates in the first week and later, due to exhaustion of proliferation area, there was a drastic reduction in their viability. Exactly the opposite was seen with C2C12 cells seeded on the THA and HA scaffolds, with a slow and suppressed proliferation in the early days followed by a gradual increase post 2-weeks. SEM analysis clearly indicated adhesion of both cell types to the materials while DAPI shows homogenous distribution of cells across the scaffolds. The levels of ALP produced by C2C12 cells seeded on the scaffolds were significantly higher than the ALP levels in the plastic controls (p<0,01) especially in the first two weeks. MSCs also had an elevated ALP level on both materials. In-vivo delivery of rhBMP-2 and ZA: Micro-CT results showed more bone formation in the THA scaffolds loaded with a combination of rhBMP-2 and ZA (p<0.01) followed by rhBMP-2 only group. Minimal or no bone formation was seen in the cryogel implanted alone. Bone formation was also confirmed histologically and typical bone morphology could be observed. Ex-vivo SEM analysis showed bone growing around the scaffold and also typical bony ossicles could be observed. EDX analysis showed more calcium deposition in rhBMP-2+ZA group compared to the others (p<0.01). Discussion Porous composite scaffolds have a huge potential in bone tissue engineering during surgically invasive procedures4. Bioactive glasses are known to enhance osteogenesis5 while HA mimics the native bone mineral. The scaffolds were characterized physico-chemically to confirm the presence of both bioactive glass and HA as well as amide linkage of chitosan and silk with glutaraldehyde. The scaffolds were stable at physiological temperatures thereby making them feasible for in-vivo usage. The scaffolds were biocompatible for both C2C12 muscle cells and rat MSC’s as seen from the MTT assay and also had an augmentation effect on the cells leading to elevated ALP levels one week post seeding. The muscle pouch model clearly indicates that THA scaffolds can be used as a carrier for both rhBMP-2 and ZA and combination of rhBMP-2 and ZA works better in this model due to anti catabolic effects of ZA. In the near future, we will assess the feasibility of using these scaffolds in relevant models of bone formation like a long bone critical defect. Significance This study describes a new and efficient carrier for the delivery of rhBMP-2 and ZA, which can avoid the high doses of BMPs administered today and reduce bone resorption by local co-delivery of bisphosphonates.

ORS 2016 Annual Meeting Paper No. 0075