Progress of Regenerative Therapy in Orthopedics.

PURPOSE OF REVIEW: To conduct a thorough appraisal of recent and inventive advances in the field of bone tissue engineering using biomaterials, cell-based research, along with the incorporation of biomimetic properties using surface modification of scaffolds. RECENT FINDINGS: This paper will provide an overview on different biomaterials and emerging techniques involved in the fabrication of scaffolds, brief description of signaling pathways involved in osteogenesis, and the effect of surface modification on the fate of progenitor cells. The current strategies used for regenerative medicine like cell therapy, gene transfer, and tissue engineering have opened numerous therapeutic avenues for the treatment of various disabling orthopedic disorders. Precise strategy utilized for the reconstruction, restoration, or repair of the bone-related tissues exploits cells, biomaterials, morphogenetic signals, and appropriate mechanical environment to provide the basic constituents required for creating new tissue. Combining all the above strategies in clinical trials would pave the way for successful "bench to bedside" transformation in bone healing.

Comparison of pregabalin with gabapentin as preemptive analgesic in lumbar spine surgery.

BACKGROUND AND AIMS: Analgesic effect of gabapentin and pregabalin is well-defined in the treatment of neuropathic pain. Postoperative pain after lumbar spine surgery limits the function of patients in the postoperative period, for which the search for ideal analgesic goes on. The aim of the present study was to compare pregabalin and gabapentin as a pre-emptive analgesic in elective lumbar spine surgeries. MATERIAL AND METHODS: In this randomized prospective study, 75 patients were allocated into three groups of 25 each. Group G, group PG, and group P received two capsules of gabapentin 300 mg each, two capsules of pregabalin 150 mg each, and two multivitamin capsules, respectively, with sip of water 1 hour before the expected time of induction of anesthesia. Time for requirement of first dose of rescue analgesia, reduction in postoperative pain score and total dose of rescue analgesic used in first 24 hours postoperatively, and side effects were compared. RESULT: Time for requirement of first dose of rescue analgesic in PG group was 180.12 min and in G group was 104.16 min, which was statistically significant. Both G and PG group had lower visual analogue scale (VAS) score in comparison to P group, which was statistically significant. Consumption of rescue analgesic was less in G and PG group in comparison to P group. Amount of rescue analgesic requirement were low in PG group in comparison to G group (P < 0.001). CONCLUSION: Though both study drugs had produced prolonged postoperative analgesia compared to placebo, pregabalin had better analgesic profile in postoperative period than gabapentin.

Stem Cell-Based Tissue Engineering Approaches for Diabetic Foot Ulcer: a Review from Mechanism to Clinical Trial.

Diabetic foot ulcer (DFU) is a complication from incomplete or prolonged wound healing, at times requires amputation, putting substantial health and socioeconomic burden. Wound healing is a dynamic overlapping process that can be regulated by arrays of molecular factors showing redundancy in function. However, dysregulation in the mechanism of angiogenesis, extra cellular matrix (ECM) formation and immune modulation are the major causes for impair wound healing in hyperglycaemic patients. Despite development of wound care research, there is a lack of well-accepted targeted therapy with multidisciplinary approach for DFU treatment. Stem cell therapy holds a promising outcome both in preclinical and clinical trials because of its ability to promote healing via regeneration and specialized tissue differentiation. Among different types of stem cells, regenerative potential of mesenchymal stem cell (MSC) is well demonstrated in both experimental and clinical trial. Still there is a huge knowledge gap among medical practitioners for deciding the best stem cell source, administration route, and safety. This review strengthens the fact that why stem cell therapy is a promising candidate to treat DFU and cited multiple tissue engineering and biomaterial-based approaches for delivering stem cells and their aftermath paracrine events. Based on the pre-clinical and clinical studies, the review tried to come up with optimum stem cell source and delivery route for the treatment of DFU. At last, the review glances on possible direction to enhance therapeutics strategy for the same, including different approaches like: phytocompounds, exosomes, scaffold geometry, cell preconditioning and licensing etc.

Artificial Intelligence-Based 3D Printing Strategies for Bone Scaffold Fabrication and Its Application in Preclinical and Clinical Investigations.

3D printing has become increasingly popular in the field of bone tissue engineering. However, the mechanical properties, biocompatibility, and porosity of the 3D printed bone scaffolds are major requirements for tissue regeneration and implantation as well. Designing the scaffold architecture in accordance with the need to create better mechanical and biological stimuli is necessary to achieve unique scaffold properties. To accomplish this, different 3D designing strategies can be utilized with the help of the scaffold design library and artificial intelligence (AI). The implementation of AI to assist the 3D printing process can enable it to predict, adapt, and control the parameters on its own, which lowers the risk of errors. This Review emphasizes 3D design and fabrication of bone scaffold using different materials and the use of AI-aided 3D printing strategies. Also, the adaption of AI to 3D printing helps to develop patient-specific scaffolds based on different requirements, thus providing feedback and adequate data for reproducibility, which can be improvised in the future. These printed scaffolds can also serve as an alternative to preclinical animal test models to cut costs and prevent immunological interference.

Mechanical and biological characteristics of 3D fabricated clay mineral and bioceramic composite scaffold for bone tissue applications.

3D printing technology provides a platform to fabricate a wide range of structures and complex geometry-based scaffolds through computer-aided design (CAD). This study investigates the possibility of developing Bentonite(BEN)/Hydroxyapatite(HAP) scaffold with different HAP wt% (25, 50, 75) using a 3D printing technique (Robocasting) for potential bone tissue applications. Thermal stability of the composites was characterized in TGA and rheological properties of slurries were observed to have different viscosity and shear stress, especially BEN-HAP 50 wt% achieves all criteria for high-quality printing. The fabricated scaffolds were subjected to sintering from 200 °C to 1000 °C for proper densification and attained a maximum compression strength of 52 MPa at 1000 °C for the printed structures. Changes in crystallinity and functional groups were observed as well with respective sintering temperatures. In this study, we also discussed the extrusion and rheological properties of the composite slurry. Porosity, water absorption, degradation and density were studied to understand the physical properties of the sintered scaffolds. The biological characteristics of the scaffold were studied using MG63 cell lines In vitro biocompatibility study and expressed 91% of viability for the 1000 °C sintered samples under controlled culture conditions.

Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering.

Silk sericin protein is a natural, hydrophilic, macromolecular glycoprotein mainly synthesized in the middle silk gland of the silkworm. It constitutes 25-30% of the silk cocoon. Sericin proteins have antioxidant, antimicrobial, UV-resistant properties, promote wound healing and support cell proliferation even in serum-free media. Most of the sericin is discarded as waste in silk processing industries. This study aims at improving the mechanical strength and stability of sericin extracted from the silk cocoons during processing and utilize it as a biocompatible natural biopolymer in biomedical applications. Crosslinked sericin membranes, from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, were prepared using gluteraldehyde as the crosslinking agent. Physical and structural characteristics of the membranes were analyzed using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and X-ray diffraction along with swelling and degradation studies. The secondary structure of the membrane indicates that crosslinking provides a more integrated structure that significantly improves the stability and mechanical strength of the membranes. In vitro cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis of feline fibroblast cells. The adherence, growth and proliferation patterns of cells on membranes were assessed by confocal microscopy, which demonstrated that the latter is non-toxic and supports cell growth. Cell cycle analyses indicate cytocompatibility with normal cell cycle pattern. This study reveals that silk sericin protein can be used as a biocompatible natural biopolymer for various applications in the biomedical field.

Biological and mechanical response of laser shock peening orthopaedic titanium alloy (Ti-6Al-7Nb).

This paper focuses on the evaluation of mechanical and biological properties of laser shock peening (LSP) orthopaedic grade Ti-6Al-7Nb alloy. LSP surface treatment was conducted at laser energy of 3 to 7 J with overlaps of 33%-67%, and with a 3 mm laser spot size. Cell viability on laser shock peened surface was evaluated through in-vitro MTT assay, using osteoblast-like MG63 cells for the first-time. Residual stresses, microhardness, microstructure, sliding wear and wetting properties were investigated. Compressive residual stresses were found at various depths due to controlling the LSP parameters, compared to the as-received surface. The laser shock peened surfaces were hardened from 365HV0.05 to 405HV0.05, while the as-received surface was 320HV0.05. The average sub-grain size was refined from 14% to 36% after LSP. The wear resistance was also controllable by altering LSP parameters. The MTT results show that the cell viability on the laser shock peened surfaces was comparatively lower than that of the untreated surface after 24 h. However, after 72 h, the cell viability on modified surfaces were significantly improved. This work indicated that laser shock peened surfaces have a strong potential to decrease the pain from orthopaedic implant failures and promote the cytocompatibility between the bone and implant.

Distribution-based dimensionality reduction applied to articulated motion recognition.

Some articulated motion representations rely on frame-wise abstractions of the statistical distribution of low-level features such as orientation, color, or relational distributions. As configuration among parts changes with articulated motion, the distribution changes, tracing a trajectory in the latent space of distributions, which we call the configuration space. These trajectories can then be used for recognition using standard techniques such as dynamic time warping. The core theory in this paper concerns embedding the frame-wise distributions, which can be looked upon as probability functions, into a low-dimensional space so that we can estimate various meaningful probabilistic distances such as the Chernoff, Bhattacharya, Matusita, Kullback-Leibler (KL) or symmetric-KL distances based on dot products between points in this space. Apart from computational advantages, this representation also affords speed-normalized matching of motion signatures. Speed normalized representations can be formed by interpolating the configuration trajectories along their arc lengths, without using any knowledge of the temporal scale variations between the sequences. We experiment with five different probabilistic distance measures and show the usefulness of the representation in three different contexts-sign recognition (with large number of possible classes), gesture recognition (with person variations), and classification of human-human interaction sequences (with segmentation problems). We find the importance of using the right distance measure for each situation. The low-dimensional embedding makes matching two to three times faster, while achieving recognition accuracies that are close to those obtained without using a low-dimensional embedding. We also empirically establish the robustness of the representation with respect to low-level parameters, embedding parameters, and temporal-scale parameters.

Suture materials - Current and emerging trends.

Surgical sutures are used to facilitate closure and healing of surgical- or trauma-induced wounds by upholding tissues together to facilitate healing process. There is a wide range of suture materials for medical purpose and the main types include absorbable and nonabsorbable. Recently, there is a growth in the development of classes of suture materials based on their properties and capabilities to improve tissue approximation and wound closure. This review outlines and discusses the current and emerging trends in suture technology including knotless barbed sutures, antimicrobial sutures, bio-active sutures such as drug-eluting and stem cells seeded sutures, and smart sutures including elastic, and electronic sutures. These newer strategies expand the versatility of sutures from being used as just a physical entity approximating opposing tissues to a more biologically active component enabling delivery of drugs and cells to the desired site with immense application potential in both therapeutics and diagnostics. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1544-1559, 2016.

Sericin-carboxymethyl cellulose porous matrices as cellular wound dressing material.

In this study, porous three-dimensional (3D) hydrogel matrices are fabricated composed of silk cocoon protein sericin of non-mulberry silkworm Antheraea mylitta and carboxymethyl cellulose. The matrices are prepared via freeze-drying technique followed by dual cross-linking with glutaraldehyde and aluminum chloride. The microstructure of the hydrogel matrices is assessed using scanning electron microscopy and biophysical characterization are carried out using Fourier transform infrared spectroscopy and X-ray diffraction. The transforming growth factor ß1 release from the cross-linked matrices as a growth factor is evaluated by immunosorbent assay. Live dead assay and 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide assay show no cytotoxicity of blended matrices toward human keratinocytes. The matrices support the cell attachment and proliferation of human keratinocytes as observed through scanning electron microscope and confocal images. Gelatin zymography demonstrates the low levels of matrix metalloproteinase 2 (MMP-2) and insignificant amount of MMP-9 in the culture media of cell seeded matrices. Low inflammatory response of the matrices is indicated through tumor necrosis factor alpha release assay. The results indicate that the fabricated matrices constitute 3D cell-interactive environment for tissue engineering applications and its potential use as a future cellular biological wound dressing material.

Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions.

The encapsulation based technology permits long-term delivery of desired therapeutic products in local regions of body without the need of immunosuppressant drugs. In this study microcapsules composed of sericin and alginate micro bead as inner core and with an outer chitosan shell are prepared. This work is proposed for live cell encapsulation for potential therapeutic applications. The sericin protein is obtained from cocoons of non-mulberry silkworm Antheraea mylitta. The sericin-alginate micro beads are prepared via ionotropic gelation under high applied voltage. The beads further coated with chitosan and crosslinked with genipin. The microcapsules developed are nearly spherical in shape with smooth surface morphology. Alamar blue assay and confocal microscopy indicate high cell viability and uniform encapsulated cell distribution within the sericin-alginate-chitosan microcapsules indicating that the microcapsules maintain favourable microenvironment for the cells. The functional analysis of encapsulated cells demonstrates that the glucose consumption, urea secretion rate and intracellular albumin content increased in the microcapsules. The study suggests that the developed sericin-alginate-chitosan microcapsule contributes towards the development of cell encapsulation model. It also offers to generate enriched population of metabolically and functionally active cells for the future therapeutics especially for hepatocytes transplantation in acute liver failure.

Non-mulberry silk fibroin influence osteogenesis and osteoblast-macrophage cross talk on titanium based surface.

The titanium and its alloys are used as orthopedic dental implants due to their mechanical and bio-inert properties. The bare metal implants are not the ultimate answer for better osteogenesis and implant integration. Physical and chemical modifications are carried out to achieve the goal of improved adhesion and differentiation of the osteoblast. In this work, the silk fibroins from both mulberry and non-mulberry sources are used for surface modification. Silk fibroins are immobilized on titanium surface to facilitate the initial cell adhesion followed by improved cell spreading and better mineralization in order to achieve enhanced osseointegration. The immunological responses along with the effect of cytokines on osteoblast adhesion and function are investigated. The non-mulberry fibroin performs better in the context of the cell adherence and differentiation, which lead to better mineralization. The results indicate that the silk fibroin from non-mulberry source can be used for better osteogenesis on orthopedic implants.

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-ß, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1ß and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

The promotion of osseointegration of titanium surfaces by coating with silk protein sericin.

A promising strategy to influence the osseointegration process around orthopaedic titanium implants is the immobilization of bioactive molecules. This recruits appropriate interaction between the surface and the tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In this study, we aimed to investigate the functionalization of metallic implant titanium with silk protein sericin. Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde as crosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugated with integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titanium was characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD functionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion, proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNA expressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblast cells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significant amount of pro-inflammatory cytokines TNF-α, IL-1ß and nitric oxide production were recorded when macrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized titanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially useful bioactive coated materials for titanium-based medical implants.

Silk hydrogels from non-mulberry and mulberry silkworm cocoons processed with ionic liquids.

Matrices based on silk fibroin from the non-mulberry silkworm Antheraea mylitta and the mulberry silkworm Bombyx mori have demonstrated good applicability in regenerative medicine. However, the cocoons of A. mylitta are underutilized in part due to their lack of solubility in traditional organic solvents. Therefore, the present work investigates the solubilization and processing of degummed fibers obtained from the cocoons of both silkworm species into hydrogels using ionic liquids (ILs). The developed hydrogels exhibited a rubbery consistency, viscoelastic behavior and rapid degradation in the presence of protease XIV. Scanning electron and confocal microscopy images suggest that human adipose stem cells (hASCs) are able to adhere to and migrate at different levels within the hydrogel structures. Moreover, the MTS assay demonstrated the maintenance of cell metabolic activity for up to 28 days, while DNA quantification showed that hASCs were able to proliferate on the seeded hydrogels. The findings indicate that complete IL removal from the fabricated hydrogels results in a positive hASCs cellular response. Thus the present approach provides a unique opportunity to broaden the processability and application of silk fibroin obtained from A. mylitta cocoons for regenerative medicine, namely cartilage regeneration.

Invited review nonmulberry silk biopolymers.

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.