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Bone regeneration therapies developed using interdisciplinary tissue engineering approaches are used to develop bio-inspired new generation materials and strategies to augment bone formation and decrease dependence on bone grafts. Due to their better mimicking of biological properties of the native tissue, these new generation synthetic bone graft substitutes can lead to enhanced bone formation and rapid defect healing in short time. These bioinspired materials can act as bridge between bone autografts and bio-inactive synthetic bone cements.Bone morphogenetic proteins (BMPs) are potent osteoinductive molecules, and are commonly used in clinics to get enhanced bone formation. However, BMPs can’t be delivered systemically and hence need to be delivered locally. For that biphasic nanocement (NC) using nanoscale hydroxyapatite (nHAP) and calcium sulphate hemihydrate is successfully employed. Various endogenous bioactive molecules are being utilized in addition to injectable substitutes and cryogels.
Nano-cement as a carrier of bioactive factors and antibiotics
Osteoporosis is a prevalent skeletal disorder and the most common cause of debilitating hip fractures, primarily affecting post-menopausal women and elderly people. Implants used to stabilize osteoporotic fragility fractures are susceptible to bacterial infections, which further deteriorate the implantation site. In the current study, we developed a nanocement-based treatment for tackling hip implant infection in an osteoporotic rat model. The development of osteoporosis was confirmed by an 80% decrease in serum estradiol levels and a further reduced T-score value of −4.170 ± 0.411 for femur neck canal. Further, an infected K-wire was implanted in femoral neck canal to induce implant-associated osteomyelitis. In order to combat infection and promote bone healing, it was followed by debridement and implantation of rifampicin-loaded nanocement functionalized with various combinations of bone marrow MSCs (bMSC) exosomes, bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA). The findings of this study demonstrated the potential of exosomes as an alternative for reducing BMP-2 dosage, and the treatment promoted successful bone regeneration with enhanced mechanical strength, and a significant decrease in bacterial load. Improved bone remodeling and matrix deposition at the implantation site were confirmed by DXA, micro-CT, and histology. Further, immunohistochemical staining showed increased expression of collagen I and CD31, which indicated bone matrix deposition and vascularization, respectively, while reduced expression of α-SMA and NOS2 confirmed a reduction in fibrosis and inflammation, respectively. Overall, our one-step treatment strategy has the potential to prevent implant-associated bone infection in osteoporotic hip fractures, promote bone formation, and enhance the mechanical strength of osteoporotic bone, thereby preventing subsequent secondary fractures.
Bone substitutes for efficient bone regeneration
Bone regeneration using gelatin-modified bone substitute with bioactive molecules
Injectable bone cement was synthesized and incorporated with gelatin to enhance cellular interaction. Human osteosarcoma Saos-2 cells derived bone morphogenetic proteins (BMP’s) and zoledronic acid were also incorporated to cement. The efficacy of these bioactive molecules in enhancing bone substitution qualities of bone cements for local delivery was investigated by implanting in 3.5 mm critical size defect in tibial metaphysis of wistar rats . 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 BMP’s compared to other groups.
■ Teotia, A. K., Gupta, A., Raina, D. B., Lidgren, L., & Kumar, A. (2016). Gelatin-modified bone substitute with bioactive molecules enhance cellular interactions and bone regeneration. ACS applied materials & interfaces, 8(17), 10775-10787.
■ Teotia, A. K., Gupta, A., Raina, D. B., Lidgren, L., & Kumar, A. (2016). Gelatin-modified bone substitute with bioactive molecules enhance cellular interactions and bone regeneration. ACS applied materials & interfaces, 8(17), 10775-10787.
Cranial bone regeneration emplyoing nano-Hydroxyapatite bone substitute functionalized with bone active molecules
Nano-hydroxyapatite (nHAP) and calcium sulfate bone substitute (NC) were synthesized and characterized for cranioplasty. The NC was functionalized with low concentrations of bone morphogenetic protein-2 (BMP2) and zoledronic acid (ZA) and characterized both in vitro and in vivo. 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.
■ Teotia, A. K., Raina, D. B., Singh, C., Sinha, N., Isaksson, H., Tägil, M., Lidgren, L. & Kumar, A. (2017). Nano-hydroxyapatite bone substitute functionalized with bone active molecules for enhanced cranial bone regeneration. ACS applied materials & interfaces, 9(8), 6816-6828.
■ Teotia, A. K., Raina, D. B., Singh, C., Sinha, N., Isaksson, H., Tägil, M., Lidgren, L. & Kumar, A. (2017). Nano-hydroxyapatite bone substitute functionalized with bone active molecules for enhanced cranial bone regeneration. ACS applied materials & interfaces, 9(8), 6816-6828.
Bone Infections
Musculoskeletal infections involving bones, muscles and joints are one of the major reasons of morbidity and mortality especially in children worldwide. A major cause for bone and joint infections is haematogenous dissemination wherein the pathogen accumulates at the site via bloodstream. Most commonly bone and joint infections is clinically manifested as osteomyelitis or bone tuberculosis. In developing countries like India, bone tuberculosis is an insidious problem affecting a large proportion of population. Bone tuberculosis is the most common form of all extra-pulmonary tuberculosis mostly affecting vertebrae, metaphyseal bone and joints accompanied by insidious pain at rest, spinal deformity with neurological impairments and obliteration of bone and cartilage. Parenteral delivery accompanying surgical interventions of antimicrobial agents has been a common approach as a treatment strategy in bone infections including bone tuberculosis. Owing to the limitations like less or no bioavailability at bone nidus site, degradation of antibiotics in acidic environment of stomach, antibiotic dilution in blood etc. has necessitated it to use an alternate approach of drug delivery system to deliver drugs at the local site of infection. Our musculoskeletal group works on design and development of a potent delivery system using indigenously synthesised nano-hydroxyapatite based ceramic as a carrier to deliver the drugs at the infection/nidus site. Our paramount goal to circumvent the limitations of conventional therapy of parenteral drug administration and develop a system that will deliver the first line anti-tuberculosis drugs Rifampicin and Isoniazid in a controlled manner and at the same time promote the regeneration of lost bone.
Osteo-chondral tissue engineering
Evaluating potential of tissue-engineered cryogels and chondrocyte derived exosomes in articular cartilage repair
Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.
Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.
Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.
In Vitro Neo‐Cartilage Formation on a Three‐Dimensional Composite Polymeric Cryogel Matrix
A novel way of fabrication, cryogelation, is presented, in which matrices are synthesized at sub-zero temperature. Cell seeded scaffolds incubated under appropriate conditions result in the accumulation of matrix components on the surface of the gel in the form of neo-cartilage.Neo-cartilage exhibits similarity to native cartilage with respect to its physical, mechanical and biochemical properties. Based onthe similarities of neo-cartilage to the native cartilage, it can provide a new approach for the treatment of localised joint injuries.
A novel way of fabrication, cryogelation, is presented, in which matrices are synthesized at sub-zero temperature. Cell seeded scaffolds incubated under appropriate conditions result in the accumulation of matrix components on the surface of the gel in the form of neo-cartilage.Neo-cartilage exhibits similarity to native cartilage with respect to its physical, mechanical and biochemical properties. Based onthe similarities of neo-cartilage to the native cartilage, it can provide a new approach for the treatment of localised joint injuries.
Image analysis of CAG cryogel scaffolds. The digital image of CAG cryogel scaffolds synthesized in monolith and disc format (a),flourescent microscopic images of CAG cryogel sections stained with phalloidin-FITC (thickness 10 mm) showing interconnected porearchitecture (b). X-ray micro-CT images (c and d), 3D image of CAG (5%) cryogel (c) homogenous porosity observed by reconstructedbinarized 2D image of CAG (d). The 3D flourescent microscopy and micro-CT analysis also shows the uniform chondrocyte cells distribution on CAG matrices when stained with phalloidin-FITC and osmium tetraoxide, respectively (e and f). Image 1f is the recostitution of different scanned images taken at varied depths showing chondrocyte penetration through the scaffold.
Endogenous Platelet-Rich Plasma Supplements for Enhanced Bone Formation
Composite collagen-nanohydroxyapatite (CS) bone filler, mimicking porous architecture of trabecular bone was developed. It was functionalized with clinically available bone active agents like bone morphogenetic protein-2 (rhBMP-2) and zoledronic acid (ZA). Synergistic effects of the bone active molecules and endogenous platelet rich plasma (PRP), a source of growth factors on mineralization was investigated. Bone formation was evaluated at ectopic sites in abdominal pouch and 4.0 mm critical defect in tibia metaphysis of rats. Tissue mineralization was evaluated by micro-CT and histological analysis 12 weeks postimplantation. In vivo BMP +ZA+PRP functionalized scaffolds had higher amount (28 mm3 ) of mineralized tissue formation as compared to empty defect (20 mm3 ), suggesting that PRP can augment the osteoinductive properties of functionalized scaffolds both in vitro and in vivo. Enhanced cell infiltration and mineralization can be achieved via CS in comparison to SC, implying their use as porous bone void fillers and substitutes for autografts
■ Teotia, A. K., Qayoom, I., & Kumar, A. (2018). Endogenous Platelet-Rich Plasma Supplements/Augments Growth Factors Delivered via Porous Collagen-Nanohydroxyapatite Bone Substitute for Enhanced Bone Formation. ACS Biomaterials Science & Engineering, 5(1), 56-69.
■ Teotia, A. K., Qayoom, I., & Kumar, A. (2018). Endogenous Platelet-Rich Plasma Supplements/Augments Growth Factors Delivered via Porous Collagen-Nanohydroxyapatite Bone Substitute for Enhanced Bone Formation. ACS Biomaterials Science & Engineering, 5(1), 56-69.
Biomaterial testing:Muscle pouch was created in the muscle and the material was placed between two layers of the muscle which were closed by sutures. All animals received two THA scaffolds alone on the left side separated by a distance of 1.5 cm each. Then the animals were further divided into two more groups based on the components they carried. Six animals received two THA scaffold combined with rhBMP-2 on the right while another six animals received two scaffolds of THA loaded with a combination of rhBMP-2 and ZA on the right. Animals had free access to food and water
THA (silk-fibroin, chitosan, agarose, bioactive glass and hydroxyapatite) and HA (silk-fibroin, chitosan, agarose, and hydroxyapatite) cryogels for bone tissue engineering. A and B show digital images of THA and HA cryogels, respectively. C, E and D, F shows light microcopy and SEM images of THA and HA cryogels, respectively.
■ Raina, D. B., Isaksson, H., Teotia, A. K., Lidgren, L., Tägil, M., & Kumar, A. (2016). Biocomposite macroporous cryogels as potential carrier scaffolds for bone active agents augmenting bone regeneration. Journal of Controlled Release, 235, 365-378.
■ Raina, D. B., Isaksson, H., Teotia, A. K., Lidgren, L., Tägil, M., & Kumar, A. (2016). Biocomposite macroporous cryogels as potential carrier scaffolds for bone active agents augmenting bone regeneration. Journal of Controlled Release, 235, 365-378.
Bone generation using biphasic calcium sulphate/ hydroxyapatite carrier containing Bone Morphogenic Protein-2 and Zoledronic Acid
(1) indicates a set disc of the biphasic material with ZA bound to HA while rhBMP-2 is encapsulated between the two phases. After in-vivo implantation, the material releases sulphate, rhBMP-2 and little ZA as shown in (2). Muscle stem cells interact with rhBMP-2 via BMP receptors and a change in their phenotype occurs leading to their osteogenic differentiation. Subsequently the bone formation approaches inwards into the scaffold. Due to sulphate resorbing over time, the scaffold gets more porous and the bone formation is substantiated by rhBMP-2 as shown in (3). After a significant amount of bone is formed, RANKL-RANK (Osteoblast- Osteoclast progenitor) interaction causes osteoclastogenesis as shown in (4). However, due to the presence of ZA, osteoclastic apoptosis occurs28 leading to a preserved bone turnover (5).
■ Raina, D. B., Isaksson, H., Hettwer, W., Kumar, A., Lidgren, L., & Tägil, M. (2016). A biphasic calcium sulphate/hydroxyapatite carrier containing bone morphogenic protein-2 and zoledronic acid generates bone. Scientific reports, 6, 26033.
Bone remodelling :
Different components of basic multicellular unit (BMU) showing cells involved in various stages of bone remodelling covering resorption of old bone to deposition of new bone.Bone remodeling is a slow process with one complete cycle requiring approximately 120 days, involvingfollowing six stages:
1. Quiescence: osteoblasts transform into osteocytes and endosteal lining cells2. Activation: recruitment of osteoclasts 3. Resorption: bone resorption by osteoclasts in bone remodeling compartment4. Reversal: migration/apoptosis of osteoclasts and debris clearing by lining cells5. Bone formation: osteoid deposition by osteoblasts6. Mineralization: mineralization of deposited osteoid
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