Deepak Bushan Raina

Gelatin-Modified Bone Substitute with Bioactive Molecules Enhance Cellular Interactions and Bone Regeneration.

In this work, we have synthesized injectable bone cement incorporated with gelatin to enhance cellular interaction. Human osteosarcoma Saos-2 cells derived bone morphogenetic proteins (BMPs) and a bisphosphonate (zoledronic acid (0.2 mM)) were also incorporated to cement. In vitro studies conducted using Saos-2 demonstrated enhanced cell proliferation on gelatin (0.2%w/v) cement. The differentiation of C2C12 mouse myoblast cells into bone forming cells showed 6-fold increase in ALP levels on gelatin cement. Polymerase chain reaction (PCR) for bone biomarkers showed osteoinductive potential of gelatin cement. We investigated efficacy for local delivery of these bioactive molecules in enhancing bone substitution qualities of bone cements by implanting in 3.5 mm critical size defect in tibial metaphysis of wistar rats. The rats were sacrificed after 12 weeks and 16 weeks post implantation. X-ray, micro-CT, histology, and histomorphometry analysis were performed to check bone healing. The cement materials slowly resorbed from the defect site leaving HAP creating porous matrix providing surface for bone formation. The materials showed high biocompatibility and initial bridging was observed in all the animals but maximum bone formation was observed in animals implanted with cement incorporated with zoledronic acid followed by cement with BMPs compared to other groups.

A Biphasic Calcium Sulphate/Hydroxyapatite Carrier Containing Bone Morphogenic Protein-2 and Zoledronic Acid Generates Bone

In orthopedic surgery, large amount of diseased or injured bone routinely needs to be replaced. Autografts are mainly used but their availability is limited. Commercially available bone substitutes allow bone ingrowth but lack the capacity to induce bone formation. Thus, off-the-shelf osteoinductive bone substitutes that can replace bone grafts are required. We tested the carrier properties of a biphasic, calcium sulphate and hydroxyapatite ceramic material, containing a combination of recombinant human bone morphogenic protein-2 (rhBMP-2) to induce bone, and zoledronic acid (ZA) to delay early resorption. In-vitro, the biphasic material released 90% of rhBMP-2 and 10% of ZA in the first week. No major changes were found in the surface structure using scanning electron microscopy (SEM) or in the mechanical properties after adding rhBMP-2 or ZA. In-vivo bone formation was studied in an abdominal muscle pouch model in rats (n = 6/group). The mineralized volume was significantly higher when the biphasic material was combined with both rhBMP-2 and ZA (21.4 ± 5.5 mm3) as compared to rhBMP-2 alone (10.9 ± 2.1 mm3) when analyzed using micro computed tomography (μ-CT) (p < 0.01). In the clinical setting, the biphasic material combined with both rhBMP-2 and ZA can potentially regenerate large volumes of bone.

Study of in Vitro and in Vivo Bone Formation in Composite Cryogels and the Influence of Electrical Stimulation.

This work studies osteoinduction and bone conduction in polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogels along with the synergistic effect of electrical stimulation. In vitro osteoinduction of C2C12 myoblast towards osteogenic lineage is demonstrated through alkaline phosphatase assay, scanning electron microscopy and energy dispersive X-ray spectroscopy. These results were followed by in vivo implantation studies of PTAC biocomposite cryogel scaffolds in the bone conduction chamber model depicting bone formation after 24 days based on immunohistological staining for osteogenic markers, i.e., collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP). Further, osteogenic differentiation of murine mesenchymal stem cells was studied with and without electrical stimulation. The q-PCR analysis shows that the electrically stimulated cryogels exhibit ~ 6 folds higher collagen type I and ~ 10 folds higher osteopontin mRNA level, in comparison to the unstimulated cryogels. Thus, PTAC biocomposite cryogels present osteoinductive and osteoconductive properties during in vitro and in vivo studies and support osteogenic differentiation of mesenchymal stem cells under the influence of electrical stimulation.

Nano-Hydroxyapatite Bone Substitute Functionalized with Bone Active Molecules for Enhanced Cranial Bone Regeneration

The aim of this study was to synthesize and characterize a nano-hydroxyapatite (nHAP) and calcium sulfate bone substitute (NC) for cranioplasty. The NC was functionalized with low concentrations of bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA) and characterized both in vitro and in vivo. In vitro studies included MTT, ALP assays, and fluorescent staining of Saos-2 (human osteoblasts) and MC3T3-E1 (murine preosteoblasts) cells cultured on NC. An in vivo study divided 20 male Wistar rats into four groups: control (defect only), NC, NC + ZA, and NC + ZA + rhBMP-2. The materials were implanted in an 8.5 mm critical size defect in the calvarium for 12 weeks. Micro-CT quantitative analysis was carried out in vivo at 8 weeks and ex vivo after 12 weeks. Mineralization was highest in the NC + ZA + rhBMP-2 group (13.0 ± 2.8 mm3) compared to the NC + ZA group (9.0 ± 3.2 mm3), NC group (6.4 ± 1.9 mm3), and control group (3.4 ± 1.0 mm3) after 12 weeks. Histological and spectroscopic analysis of the defect site provided a qualitative confirmation of neo-bone, which was in agreement with the micro-CT results. In conclusion, NC can be used as a carrier for bioactive molecules, and functionalization with rhBMP-2 and ZA in low doses enhances bone regeneration.

Biocomposite macroporous cryogels as potential carrier scaffolds for bone active agents augmenting bone regeneration.

Osteoinduction can be enhanced by combining scaffolds with bone morphogenic protein-2 (BMP-2). However, BMPs are known to also cause bone resorption. This can be controlled using bisphosphonates like zoledronic acid (ZA). In this study, we produced two different scaffolds containing silk-fibroin, chitosan, agarose and hydroxyapatite (HA) with and without bioactive glass. The aims of the study were to fabricate, physico-chemically characterize and evaluate the carrier properties of the scaffolds for recombinant human BMP-2 (rhBMP-2) and ZA. Scaffolds were characterized using various methods to confirm their composition. During cell-material interactions, both scaffolds exhibited gradual but sustained proliferation of both C2C12 and MSCs for a period of 6weeks with augmentative effects on their phenotype indicated by elevated levels of alkaline phosphatase (ALP) cuing towards osteogenic differentiation. In-vitro effects of rhBMP-2 and ZA contained within both the scaffolds was assessed on MC3T3 preosteoblast cells and the results show a significant increase in the ALP activity of the cells seeded on scaffolds with rhBMP-2. Further, the scaffold with both HA and bioactive glass was considered for the animal study. In-vitro, this scaffold released nearly 25% rhBMP-2 in 21-days and the addition of ZA did not affect the release. In the animal study, the scaffolds were combined with rhBMP-2 and ZA, rhBMP-2 or implanted alone in an ectopic muscle pouch model. Significantly higher bone formation was observed in the scaffold loaded with both rhBMP-2 and ZA as seen from micro-computed tomography, histomorphometry and energy dispersive X-ray spectroscopy.

Fabrication temperature modulates bulk properties of polymeric gels synthesized by different crosslinking methods

The effects of a natural matrix like extracellular matrix (ECM) on cell growth, proliferation and induction of mechanotransduction signals are well documented. Mimicking the mechanical and rheological character of ECM to repair, reconstruct and regenerate injured or impaired tissue using the concepts of tissue engineering is of significant importance. We intended to study the effect of “synthesis temperature” on mechanical and rheological properties of monomeric and polymeric scaffolds fabricated from either monomer based polyacrylamide gels or by physical gelation (agarose gels) or chemical crosslinking (chitosan, chitosan–gelatin and chitosan–agarose–gelatin (CAG) gels) at −15 °C, −5 °C or 25 °C. Our results indicated that in the absence of porogens in the form of ice crystals, conventional hydrogels had higher mechanical strength and low porosity as compared to cryogels synthesized at −15 °C and −5 °C. Furthermore, rheological analysis performed on these gels indicated temperature dependence of rheology. CAG cryogels had higher storage modulus as compared to freeze dried (FD) hydrogels or conventional hydrogels in the wet state making them amiable for cartilage tissue regeneration. Out of all synthesized gels, CAG showed better mechanical and rheological properties and was further studied to analyze the interaction with primary chicken chondrocytes. In vitro experiments on CAG scaffolds revealed a significant amount of ECM production along with good cell proliferation as indicated by MTT assay and SEM analysis.

Gelatin- hydroxyapatite- calcium sulphate based biomaterial for long term sustained delivery of bone morphogenic protein-2 and zoledronic acid for increased bone formation : In-vitro and in-vivo carrier properties

ABSTRACT In this study, a novel macroporous composite biomaterial consisting of gelatin‐hydroxyapatite‐calcium sulphate for delivery of bone morphogenic protein‐2 (rhBMP‐2) and zoledronic acid (ZA) has been developed. The biomaterial scaffold has a porous structure and functionalization of the scaffold with rhBMP‐2 induces osteogenic differentiation of MC3T3‐e1 cells seen by a significant increase in biochemical and genetic markers of osteoblastic differentiation. In‐vivo muscle pouch experiments showed higher mineralization using scaffold + rhBMP‐2 when compared to an approved absorbable collagen sponge (ACS) + rhBMP‐2 as verified by micro‐CT. Co‐delivery of rhBMP‐2 + ZA via the novel scaffold enabled a reduction in the effective rhBMP‐2 doses. The presence of tartrate resistant acid phosphatase staining in the rhBMP‐2 group indicates osteoclastic resorption, which could be stalled by adding ZA, which by speculation could explain the net increase in mineralization. The new scaffold allowed for slow release of rhBMP‐2 in‐vitro (3.3 ± 0.1%) after 4 weeks. Using single photon emission computed tomography (SPECT), the release kinetics of 125I–rhBMP‐2 in‐vivo was followed for 4 weeks and a total of 65.3 ± 15.2% 125I–rhBMP‐2 was released from the scaffolds. In‐vitro 14C–ZA release curve shows an initial burst release on day 1 (8.8 ± 0.7%) followed by a slow release during the following 4 weeks (13 ± 0.1%). In‐vivo, an initial release of 43.2 ± 7.6% of 14C–ZA was detected after 1 day, after which the scaffold retained the remaining ZA during 4‐weeks. Taken together, our results show that the developed biomaterial is an efficient carrier for spatio‐temporal delivery of rhBMP‐2 and ZA leading to increased bone formation compared to commercially available carrier for rhBMP‐2. Graphical abstract Figure. No Caption available.

Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial

Objectives We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (sd) 0.8) and BMP-7 (50.6 ng/mg, sd 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1.

Calcium Sulphate/Hydroxyapatite Carrier for Bone Formation in the Femoral Neck of Osteoporotic Rats

This study investigated bone regeneration in the femoral neck canal of osteoporotic rats using a novel animal model. A calcium sulphate (CS)/hydroxyapatite (HA) carrier was used to deliver a bisphosphonate, zoledronic acid (ZA), locally, with or without added recombinant human bone morphogenic protein-2 (rhBMP-2). Twenty-eight-week-old ovariectomized Sprague–Dawley rats were used. A 1 mm diameter and 8 mm long defect was created in the femoral neck by drilling from the lateral cortex in the axis of the femoral neck, leaving the surrounding cortex intact. Three treatment groups and one control group were used: (1) CS/HA alone, (2) CS/HA + ZA (10 μg) (3) CS/HA + ZA (10 μg) + rhBMP-2 (4 μg), and (4) empty defect (control). The bone formation was assessed at 4 weeks post surgery using in vivo micro computed tomography (micro-CT). At 8 weeks post surgery, the animals were sacrificed, and both defect and contralateral femurs were subjected to micro-CT, mechanical testing, and histology. Micro-CT results showed that the combination of CS/HA with ZA or ZA + rhBMP-2 increased the bone formation in the defect when compared to the other groups and to the contralateral hips. Evidence of new dense bone formation in CS/HA + ZA and CS/HA + ZA + rhBMP-2 groups was seen histologically. Mechanical testing results showed no differences in the load to fracture between the treatments in either of the treated or contralateral legs. The CS/HA biomaterial can be used as a carrier for ZA and rhBMP-2 to regenerate bone in the femoral neck canal of osteoporotic rats.

Cryogels and Related Research: A Glance over the Past Few Decades

This chapter describes how cryogels have evolved as promising matrices in various biotechnological and biomedical application areas. Cryogels date back nearly 40 years and here we describe the origin of these materials followed by characterization and their applications and also emphasize the differences in cryogelation and other related techniques. The main aim is to familiarize readers with the principles underlying the mechanism of cryogelation, how these matrices are characterized, provide historical and statistical data on their evolution over the past few decades and finally provide a brief idea of their applications in the areas of separation sciences, tissue engineering, environmental sciences, and so on.