Browsing by Author "Nair, RP"

Now showing 1 - 9 of 9
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    Contribution of fibroblasts to the mechanical stability of in vitro engineered dermal-like tissue through extra cellular matrix deposition
    (Bio Research Open Access, 2014-10) Nair, RP; Joseph, J; Harikrishnan, VS; Krishnan, VK; Krishnan, LK
    Tissue-engineered skin with mechanical and biological properties that match the native tissue could be a valuable graft to treat non-healing chronic wounds. Fibroblasts grown on a suitable biodegradable scaffold are a feasible strategy for the development of a dermal substitute above which epithelialization may occur naturally. Cell growth and phenotype maintenance are crucial to ensure the functional status of engineered tissue. In this study, an electrospun biodegradable polymer scaffold composed of a terpolymer PLGC [poly(lactide-glycolide- caprolactone)] with appropriate mechanical strength was used as a scaffold so that undesirable contraction of the wound could be prevented when it was implanted. To enhance cell growth, synthetic PLGC was incorporated with a fibrin-based biomimetic composite. The efficacy of the hybrid scaffold was evaluated by comparing it with bare PLGC in terms of fibroblast growth potential, extracellular matrix (ECM) deposition, polymer degradation, and mechanical strength. A significant increase was observed in fibroblast attachment, proliferation, and deposition of ECM proteins such as collagen and elastin in the hybrid scaffold. After growing fibroblasts for 20 d and 40 d, immunochemical staining of the decellularized scaffolds showed deposition of insoluble collagen and elastin on the hybrid scaffold but not on the bare scaffold. The loss of mechanical strength consequent to in vitro polymer degradation seemed to be balanced owing to the ECMdeposition. Thus, tensile strength and elongation were better when cells were grown on the hybrid scaffold rather than the bare samples immersed in culture medium. Similar patterns of in vivo and in vitro degradation were observed during subcutaneous implantation and fibroblast culture, respectively. We therefore postulate that a hybrid scaffold comprising PLGC and fibrin is a potential candidate for the engineering of dermal tissue to be used in the regeneration of chronic wounds.
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    Feasibility of a dermal substitute construction on hybrid scaffold made of poly(ε- caprolactone) and bio mimetic fibrin composite
    (Journal of Biomaterials and Tissue Engineering, 2014-09) Nair, RP; Krishnan, VK; Krishnan, LK
    Non-healing wounds can be a major problem in diabetic and burn victims. Identified causes of chronic wound formation include angiopathy, neuropathy, infection and loss of extra cellular matrix (ECM) due to increased protease action. Currently available conventional therapy is not efficient enough to promote wound healing. Tissue-engineered skin substitutes are now considered a better strategy for chronic wound management. Non-toxic scaffolds on which cells grow and replace the lost ECM are important components for tissue engineering. Degradable polymers are preferred because they may be absorbed by the time the tissue regenerate. We attempted to fabricate a degradable membrane-like and porous poly(ε-caprolactone)(PCL) scaffold to favour in-growth of cells, penetration of nutrients and oxygen. Modifying porous PCL membrane using a fibrin-based biomimetic matrix could promote cell attachment and growth of tissue. The benefit of such a hybrid scaffold for long-term cell growth and ECM deposition, parallel to the degradation of polymer and diminution of mechanical property, was evaluated. We concluded that the hybrid scaffold we developed is suitable for dermal tissue construction in vitro. When grown on the hybrid scaffold, cells synthesized and deposited insoluble ECM proteins, thus proposing better epithelialization and angiogenesis when such dermal tissue constructs are implanted for treating non-healing chronic wounds.
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    Feasibility of Dermal Substitute Construction on Hybrid Scaffold Made of Poly(epsilon-caprolactone) and Bio Mimetic Fibrin Composite
    (JOURNAL OF BIOMATERIALS AND TISSUE ENGINEERING, 2014) Nair, RP; Krishnan, K; Krishnan, L
    Non-healing wounds can be a major problem in diabetic and burn victims. Identified causes of chronic wound formation include angiopathy, neuropathy, infection and loss of extra cellular matrix (ECM) due to increased protease action. Currently available conventional therapy is not efficient enough to promote wound healing. Tissue-engineered skin substitutes are now considered a better strategy for chronic wound management. Non-toxic scaffolds on which cells grow and replace the lost ECM are important components for tissue engineering. Degradable polymers are preferred because they may be absorbed by the time the tissue regenerate. We attempted to fabricate a degradable membrane-like and porous poly(epsilon-caprolactone)(PCL) scaffold to favour in-growth of cells, penetration of nutrients and oxygen. Modifying porous PCL membrane using a fibrin-based bionnimetic matrix could promote cell attachment and growth of tissue. The benefit of such a hybrid scaffold for long-term cell growth and ECM deposition, parallel to the degradation of polymer and diminution of mechanical property, was evaluated. We concluded that the hybrid scaffold we developed is suitable for dermal tissue construction in vitro. When grown on the hybrid scaffold, cells synthesized and deposited insoluble ECM proteins, thus proposing better epithelialization and angiogenesis when such dermal tissue constructs are implanted for treating non-healing chronic wounds.
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    Identification of p63(+) keratinocyte progenitor cells in circulation and their matrix-directed differentiation to epithelial cells
    (STEM CELL RESEARCH & THERAPY, 2013) Nair, RP; Krishnan, LK
    Introduction: In the event of chronic diabetes or burn wounds, accomplishing skin regeneration is a major concern. Autologous skin grafting is the most effective remedy, but the tissue harvest may create more nonhealing wounds. Currently available skin substitutes have a limited clinical outcome because of immune reactions arising from the xenobiotic scaffold or allogenous cells. Autologous stem cells that can be collected without an additional injury may be a viable option for skin-tissue engineering. Presence of a low number of keratinocyte progenitor cells (KPCs) within the peripheral blood mononuclear cell (PBMNC) population has been indicated. Identification, isolation, expansion, and differentiation of KPCs is necessary before they are considered for skin regeneration, which is the focus of this study. Methods: Culture of isolated human PBMNCs on a cell-specific matrix was carried out to induce differentiation of KPCs. Flow cytometry and reverse transcriptase polymerase chain reaction were done for epithelial stem cell marker p63 and lineage markers cytokeratin 5 and cytokeratin 14, to track differentiation. Proliferation was confirmed by quantifying the proliferating cell nuclear antigen-expressing cells. Immunostaining with epithelial cell markers, involucrin and filaggrin, was carried out to establish terminal differentiation. Microscopic analysis confirmed growth and survival of KPCs on the dermal fibroblast monolayer and on a transplantable fibrin sheet. Results: We demonstrated that KPCs are p63(+) and CD34(-). The specifically designed composition of the extracellular matrix was found to support selective adhesion, proliferation, and differentiation of p63(+) KPCs. The PBMNC culture for 12 days under controlled conditions resulted in a homogenous population that expressed cytokeratins, and >90% of the cells were found to proliferate. Subculture for 5 days resulted in expression of filaggrin and involucrin, suggesting terminal differentiation. Transfer of matrix-selected KPCs to a dermal fibroblast monolayer or fibrin supported cell proliferation and showed typical hexagonal morphology of keratinocytes within 15 days. Conclusions: Circulating KPCs were identified with p63, which differentiated into keratinocytes with expression of the cytokeratins, involucrin and filaggrin. Components of the specifically designed matrix favored KPC attachment, directed differentiation, and may turn out to be a potential vehicle for cell transplantation.
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    In vitro release study and antimicorbial property evaluation of ofloxacin loaded poly (2-hydroxyethyl methacrylate)/poly (caprolactone)/poly (ethylene glycol) hydrogel system for burn wound management
    (Journal of Drug Delivery and therapeutics, 2017-01) Rethikala, PK; Nair, RP; Krishnan, LK; Krishnan, VK
    Monomer 2-hydroxy ethyl methacrylate containing small amounts of poly(caprolactone) and poly(ethylene glycol) incorporated with an antibiotic ofloxacin was polymerized by photo-polymerization technique using 2,4,6 trimethyl benzoyl diphenyl phosphine oxide (TPO) as photo-initiator. Encapsulation efficiency and in vitro drug release was studied using UV-visible spectroscopy. Swelling analysis was resorted to compare fluid uptake ability of hydrogel containing the drug with bare polymer. Zone of inhibition assay showed hydrogel containing 1% Ofloxacin to possess strong antimicrobial property Hemolysis assay demonstrated the hydrogel system to be non-hemolytic. Non-cytotoxic character of the hydrogel was confirmed using fibroblast cells. Cell adhesion studies showed non-attachment of fibroblasts to the polymer and improved cell proliferation simultaneously.
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    Mechanically stable and functional dermal-like tissue generation on biodegradable PLGC-fibrin hybrid scaffold
    (BioResearch Open Access., 2015-01) Nair, RP; Joseph, J; Harikrishnan, VS; Kalliyanakrishnan, V; Krishnan, LK
    Tissue-engineered skin with mechanical and biological properties that match the native tissue could be a valuable graft to treat non-healing chronic wounds. Fibroblasts grown on a suitable biodegradable scaffold are a feasible strategy for the development of a dermal substitute above which epithelialization may occur naturally. Cell growth and phenotype maintenance are crucial to ensure the functional status of engineered tissue. In this study, an electrospun biodegradable polymer scaffold composed of a terpolymer PLGC [poly(lactide-glycolide-caprolactone)] with appropriate mechanical strength was used as a scaffold so that undesirable contraction of the wound could be prevented when it was implanted. To enhance cell growth, synthetic PLGC was incorporated with a fibrin-based biomimetic composite. The efficacy of the hybrid scaffold was evaluated by comparing it with bare PLGC in terms of fibroblast growth potential, extracellular matrix (ECM) deposition, polymer degradation, and mechanical strength. A significant increase was observed in fibroblast attachment, proliferation, and deposition of ECM proteins such as collagen and elastin in the hybrid scaffold. After growing fibroblasts for 20 d and 40 d, immunochemical staining of the decellularized scaffolds showed deposition of insoluble collagen and elastin on the hybrid scaffold but not on the bare scaffold. The loss of mechanical strength consequent to in vitro polymer degradation seemed to be balanced owing to the ECM deposition. Thus, tensile strength and elongation were better when cells were grown on the hybrid scaffold rather than the bare samples immersed in culture medium. Similar patterns of in vivo and in vitro degradation were observed during subcutaneous implantation and fibroblast culture, respectively. We therefore postulate that a hybrid scaffold comprising PLGC and fibrin is a potential candidate for the engineering of dermal tissue to be used in the regeneration of chronic wounds.
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    Reinforcement of amniotic membrane with fibrin coated poly- [Lactide-co-Glycolide-co-Caprolactone] terpolymer containing silver nanoparticles for potential wound healing applications
    (International Journal of Polymeric Materials and Polymeric Biomaterials, 2019-06) Ramakrishnan, R; Krishnan, LK; Nair, RP; Krishnan, KV
    Human amniotic membrane (AM) is reported to possess remarkable therapeutic potential for wound healing due to its inherent bioactive and biocompatible properties. However, poor handling characteristics have limited its clinical use on wounds to a great extent. Reinforcement of AM with a biodegradable polymer is therefore expected to provide adequate mechanical and handling nature. Poly-[Lactide-co-Glycolide-co-Caprolactone] terpolymer incorporated with silver nanoparticles and coated with fibrin is used in this study to reinforce the membrane which delivers bioactive molecules to the wound site without wound contraction. This combination scaffold exhibited desirable properties with excellent biocompatibility for potential use in wound healing applications.
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    Strontium Hydroxyapatite scaffolds engineered with stem cells aid osteointegration and osteogenesis in osteoporotic sheep model.
    (Biointerfaces, 2018-02) Chandran, S; Shenoy, SJ; Babu, SS; Nair, RP; Varma, HK; John, A
    Osteoporotic fracture healing is an orthopaedic challenge due to excessive bone resorption and impaired osteogenesis. Majority of currenttreatment strategies focus on regulating bone resorption and the potential application of Mesenchymal Stem Cells (MSCs) in promoting osteogenesis has not been explored much. Furthermore, the present study has put forth a novel approach, wherein the synergistic action of Strontium (Sr) and MSCs in a single implant may facilitate osteoporotic bone healing. Strontium Hydroxyapatite (SrHA) synthesized by wet precipitation was fabricated into tissue engineered Strontium incorporated Hydroxyapatite (cSrHA) using sheep adipose tissue derived MSCs (ADMSCs). Porosity, radiopacity and cytocompatibility of SrHA scaffolds were found appropriate for orthopaedic applications. cSrHA scaffolds exhibited an in vitro Alkaline Phosphatase activity of 20 mol pnp/30 min comparable to that of Hydroxyapatite (HA) – control scaffold, proving its osteogenic efficacy. Implantation studies in sheep osteoporotic model depicted enhanced osteogenic ability with mature lamellar bone formation in cSrHA implanted group, compared to bare HA, SrHA and tissue engineered HA implanted groups. Histomorphometry data substantiated improved osteogenesis on par with material resorption, as cSrHA implanted group exhibited highest regeneration ratio of 0.38 ± 0.05. Density histograms from micro CT further signified the enhanced osteointegrative ability of cSrHA implants. Results of the study depicted the therapeutic potential of cSrHA in osteoporotic bone healing and proposes the use of allogenic ADMSCs for fabricating “Off the Shelf Tissue Engineered Products”