Microsphere embedded hydrogel construct - binary delivery of alendronate and BMP-2 for superior bone regeneration.

Biomimetic delivery of osteoinductive growth factors via an osteoconductive matrix is an interesting approach for stimulating bone regeneration. In this context, the bone extracellular matrix (ECM) has been explored as an optimal delivery system, since it releases growth factors in a spatiotemporal manner from the matrix. However, a bone ECM hydrogel alone is weak, unstable, and prone to microbial contamination and also has been reported to have significantly reduced bone morphogenic protein-2 (BMP-2) post decellularization. In the present work, a microsphere embedded osteoinductive decellularized bone ECM/oleoyl chitosan based hydrogel construct (BOC) was developed as a matrix allowing dual delivery of an anti-resorptive drug (alendronate, ALN, via the microspheres) and BMP-2 (via the hydrogel) for a focal tibial defect in a rabbit model. The synthesized gelatin microspheres (GMs) were spherical in shape with diameter ∼32 µm as assessed by SEM analysis. The BOC construct showed sustained release of ALN and BMP-2 under the studied conditions. Interestingly, amniotic membrane-derived stem cells (HAMSCs) cultivated on the hydrogel construct demonstrated excellent biocompatibility, cell viability, and active proliferation potential. Additionally, cell differentiation on the constructs showed an elevated expression of osteogenic genes in an RT-PCR study along with enhanced mineralized matrix deposition as demonstrated by alkaline phosphatase (ALP) assay and alizarin red assay. The hydrogel construct was witnessed to have improved neo-vascularization potential in a chick chorioalantoic membrane (CAM) assay. Also, histological and computed tomographic findings evidenced enhanced bone regeneration in the group treated with the BOC/ALN/BMP hydrogel construct in a rabbit tibial defect model. To conclude, the developed multifunctional hydrogel construct acts as an osteoinductive and osteoconductive platform facilitating controlled delivery of ALN and BMP-2, essential for stimulating bone tissue regeneration.

Decellularized bone matrix/oleoyl chitosan derived supramolecular injectable hydrogel promotes efficient bone integration.

Hydrogels derived from decellularized extracellular matrix (ECM) have been widely used as a bioactive matrix for facilitating functional bone tissue regeneration. However, its poor mechanical strength and fast degradation restricts the extensive use for clinical application. Herein, we present a crosslinked decellularized bone ECM (DBM) and fatty acid modified chitosan (oleoyl chitosan, OC) based biohybrid hydrogel (DBM/OC) for delivering human amnion-derived stem cells (HAMSCs) for bone regeneration. DBM/OC hydrogel were benchmarked against collagen-I/OC (Col-I/OC) based hydrogel in terms of their morphological characteristics, rheological analysis, and biological performances. DBM/OC hydrogel with its endogenous growth factors recapitulates the nanofibrillar 3D tissue microenvironment with improved mechanical strength and also exhibited antimicrobial potential along with superior proliferation/differentiation ability. HAMSCs encapsulation potential of DBM/OC hydrogel was established by well spread cytoskeleton morphology post 14 days of cultivation. Further, ex-vivo chick chorioallantoic membrane (CAM) assay revealed excellent neovascularization potential of DBM/OC hydrogel. Subcutaneously implanted DBM/OC hydrogel did not trigger any severe immune response or infection in the host after 21 days. Also, DBM/OC hydrogels and HAMSCs encapsulated DBM/OC hydrogels were implanted at the tibial defect in a rabbit model to assess the bone regeneration ability. Quantitative micro-CT and histomorphological analysis demonstrated that HAMSCs encapsulated DBM/OC hydrogel can support more mature mineralized bone formation at the defect area compared to DBM/OC hydrogel or SHAM. These findings manifested the efficacy of DBM/OC hydrogel as a functional cell-delivery vehicle and osteoinductive template to accelerate bone regeneration.

Bioinspired 3D porous human placental derived extracellular matrix/silk fibroin sponges for accelerated bone regeneration.

Critical bone defects arising from traumatic injury and diseases are of major health concern since they are unable to heal spontaneously without clinical intervention. In this context, bone tissue engineering provides an attractive approach to treat bone defects by providing a bioactive template which has the potential to guide osseous tissue regeneration. In this study, porous hybrid placental extracellular matrix sponge (PIMS) was fabricated by a combinatorial method using silk fibroin (SF)/placental derived extracellular matrix and subsequently evaluated its efficacy towards bone tissue regeneration. The presence of intrinsic growth factors was evidenced by immunoblotting of the extracted proteins derived from the placental derived extracellular matrix. This growth factor rich PIMS lends a unique bioactive scaffolding to human amniotic mesenchymal stem cells (HAMSCs) which supported enhanced proliferation as well as superior osteogenic differentiation. Gene expression studies demonstrated significant up-regulation of osteogenic related genes in the PIMS group. PIMS when implanted in the chick chorioallantoic membrane, significantly attracted allantoic vessels revealing its potential to stimulate angiogenesis ex vivo. Furthermore, no severe immune response to the host was observed on subcutaneous implantation of PIMS in vivo. Instead, it supported the formation of blood vessels, revealing its outstanding biocompatibility. Additionally, critical tibial defects treated with PIMS demonstrated higher bone volume after six weeks when analyzed by micro-CT, which was accompanied by high mineral density. Histological and immunofluorescence studies validated the results and revealed enhanced osseous tissue regeneration after six weeks of surgery. All these findings recapitulated that the growth factors incorporated bioactive PIMS could perform as an appropriate matrix for osteogenic differentiation and efficient bone regeneration.

Influence of Porosity and Pore-Size Distribution in Ti6Al4 V Foam on Physicomechanical Properties, Osteogenesis, and Quantitative Validation of Bone Ingrowth by Micro-Computed Tomography.

Cementless fixation for orthopedic implants aims to obviate challenges associated with bone cement, providing long-term stability of bone prostheses after implantation. The application of porous titanium and its alloy-based implants is emerging for load-bearing applications due to their high specific strength, low stiffness, corrosion resistance, and superior osteoconductivity. In this study, coagulant-assisted foaming was utilized for the fabrication of porous Ti6Al4 V using egg-white foam. Samples with three different porosities of 68.3%, 75.4%, and 83.1% and average pore sizes of 92, 178, and 297 µm, respectively, were prepared and subsequently characterized for mechanical properties, osteogenesis, and tissue ingrowth. A microstructure-mechanical properties relationship study revealed that an increase of porosity from 68.3 to 83.1% increased the average pore size from 92 to 297 µm with the subsequent reduction of compresive strength by 85% and modulus by 90%. Samples with 75.4% porosity and a 178 µm average pore size produced signifcant osteogenic effects on human mesenchymal stem cells, which was further supported by immunocytochemistry and real-time polymerase chain reaction data. Quantitative assessment of bone ingrowth by micro-computed tomography revealed that there was an approximately 52% higher bone formation and more than 90% higher bone penetration at the center of femoral defects in rabbit when implanted with Ti6Al4 V foam (75.4% porosity) compared to the empty defects after 12 weeks. Hematoxylin and eosin (H&E) and Masson trichrome (MT) staining along with energy-dispersive X-ray mapping on the sections obtained from the retrieved bone samples support bone ingrowth into the implanted region.