Peripheral Blood As a Source of Stem Cells for Regenerative Medicine: Emphasis Towards Corneal Epithelial Reconstruction-An In Vitro Study.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage. Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs), other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION: PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

Bioengineered corneal epithelial cell sheet from mesenchymal stem cells-A functional alternative to limbal stem cells for ocular surface reconstruction.

Limbal stem cell deficiency (LSCD) is the loss of limbal stem cells that reside in the corneoscleral junction resulting in vision loss or blindness. Bilateral LSCD is usually treated by allogeneic corneal transplantation, with instances of tissue rejection or failure in long-term follow-up. This study aims to use adipose mesenchymal stem cells (ASC) as an alternative autologous cell source for treating bilateral limbal deficiency conditions. ASCs derived from rabbit fat tissue were differentiated into corneal epithelial lineage using limbal explant condition media. Apart from transdifferentiation, ASC sheets were developed to facilitate effective delivery of these cells to the damage site. A thermoresponsive polymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) was synthesized and characterized to demonstrate ASC sheet formation. Transdifferentiated ASCs showed positive expression of corneal epithelial marker CK3/12 on immunostaining, supported by gene expression studies. in vivo studies by transplanting cell sheet in rabbit models of corneal injury showed clear and smooth cornea in comparison to the sham models. Histology revealed a sheet of cells aligned and integrated on to the injured corneal surface, 1 month posttransplantation. Identifying ASCs as an alternative cell source along with cell sheet technology will be a novel step in the field of corneal surface therapies.

Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study.

BACKGROUND AND OBJECTIVE: Various types of osteoconductive graft materials are used for the management of alveolar bone defects arising out of periodontal disease. Inorganic, self-setting, bioactive bone cements are suggested to be most appropriate because they can conformally fill the bone defect and resorb progressively along with the regeneration of the host site. A new calcium sulfate-based bioactive bone cement (BioCaS) is developed, having simplicity and effectiveness for bone grafting applications. The response of primary human periodontal ligament (hPDL) cells to this material is investigated through in vitro cell culture model so as to qualify it for the repair of periodontal infrabony defects. METHOD: The BioCaS was designed as powder-liquid combination with in-house synthesized high purity calcium sulfate hemihydrate incorporating hydrogen orthophosphate ions. hPDL cells were isolated, cultured and characterized using optimized primary cell culture techniques. The cytotoxicity and cytocompatibility of the BioCaS samples were evaluated using the hPDL cells, with hydroxyapatite ceramic material as control. Osteogenic differentiation of the hPDL cells in presence of BioCaS was also evaluated using Alizarin red staining, Alizarin red assay, Von Kossa staining and Masson's trichrome staining. RESULTS: The primary cell culture techniques yielded a healthy population of periodontal ligament cells, with fibroblast morphology and characteristic marker expressions. The hPDL cells exhibited good viability, adhesion and spreading to the BioCaS cement in comparison to sintered hydroxyapatite. In addition, the cells differentiated to osteogenic lineage in the presence of the BioCaS cement, without extraneous osteogenic supplements, confirming the inherent bioactivity of the cement. CONCLUSION: The new BioCaS cement is a potential candidate for the repair of periodontal defects.

Direct cell imprint lithography in superconductive carbon black polymer composites: process optimization, characterization and in vitro toxicity analysis.

Cell imprint lithography (CIL) or cell replication plays a vital role in fields like biomimetic smart culture substrates, bone tissue engineering, cell guiding, cell adhesion, tissue engineering, cell microenvironments, tissue microenvironments, cell research, drug delivery, diagnostics, therapeutics and many other applications. Herein we report a new formulation of superconductive carbon black photopolymer composite and its characterization towards a CIL process technique. In this article, we demonstrated an approach of using a carbon nanoparticle-polymer composite (CPC) for patterning cells. It is observed that a 0.3 wt % load of carbon nanoparticles (CNPs) in a carbon polymer mixture (CPM) was optimal for cell-imprint replica fabrication. The electrical resistance of the 3-CPC (0.3 wt %) was reduced by 68% when compared to N-CPC (0 wt %). This method successfully replicated the single cell with sub-organelle scale. The shape of microvesicles, grooves, pores, blebs or microvilli on the cellular surface was patterned clearly. This technique delivers a free-standing cell feature substrate. In vitro evaluation of the polymer demonstrated it as an ideal candidate for biomimetic biomaterial applications. This approach also finds its application in study based on morphology, especially for drug delivery applications and for investigations based on molecular pathways.

Osteogenic apatite particles by sol-gel assisted electrospraying.

Electrospraying has tremendous potential to prepare submicron to nano size ceramic particles with novel properties. In this study, a sol-gel assisted electrospraying has been used to synthesise phase controlled apatite (hydroxyapatite, HA and calcium deficient hydroxyapatite, CDHA) particles. Variation in particle size was also achieved by controlling the process parameters. The particles were non cytotoxic, induced proliferation of osteoblast-like cells (HOS) and internalised by the cells. Increased alkaline phosphatase, collagen and calcium deposition confirmed the mineralisation of cells. Expression of osteopontin, osteocalcin and alkaline phosphatase genes further ascertained that the particles promoted osteogenic commitment of the rat bone marrow-derived mesenchymal stem cells (rBMSCs). The particles also showed better loading and release of tetracycline drug than accelerated microwave synthesised apatite particles. The methodology for synthesis of ceramic particles may have avenues for a wide range of biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1941-1954, 2018.

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

Electrospun 3D composite scaffolds for craniofacial critical size defects.

Critical size defects in the craniofacial region can be effectively treated using three dimensional (3D) composite structures mimicking natural extra cellular matrix (ECM) and incorporated with bioactive ceramics. In this study we have shown that the dynamic liquid bath collector can be used to form electrospun polycaprolactone (PCL)-hydroxyapatite (HA) composite structure as unique 3D scaffold. The structure was found to have three distinct sections (base, stem and head) based on the mechanism of its formation and morphology. The size of the head portion was around 15 mm and was found to vary with the process parameters. Scanning electron microscopy (SEM) analysis revealed that the base had random fibres while the fibres in stem and head sections were aligned but perpendicular to each other. X-ray diffraction (XRD) analysis also showed an increase in the crystallinity index of the fibres from base to head section. Cytotoxicity and cytocompatibility studies using human osteosarcoma (HOS) cells showed good cell adhesion and proliferation indicating the suitability of the 3D structure for craniofacial graft applications.

Differential expression of transcription factors NF-κB and STAT3 in periodontal ligament fibroblasts and gingiva of healthy and diseased individuals.

OBJECTIVE: Pathogens and host mediators can activate transcription factors in periodontal cells to bring about gene level alterations, thereby accentuating the periodontal disease process. Nuclear factor-kappa B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) are two pivotal transcription factors implicated in chronic inflammatory diseases. But their importance in periodontal pathogenesis has not been investigated in detail. The aim of the present study was to evaluate the expression of activated transcription factors and their target genes in healthy and diseased periodontium. DESIGN: Primary culture of periodontal ligament fibroblasts (PDLF) were established from healthy and diseased periodontium using explant culture methods. NF-κB and STAT3 activation in these cells by Porphyromonas gingivalis LPS (lipopolysaccharide) was demonstrated using confocal microscopy and mRNA expression of target genes were evaluated by quantitative real time PCR. NF-κB and STAT3 expression in diseased and healthy gingival tissues were analyzed using immunohistochemistry. RESULTS: A basal upregulation of transcription factors and their target genes were noted in diseased PDLF compared to healthy ones. LPS challenge induced differential expression of NF-κB and STAT3 and their target genes in diseased PDLF compared to healthy ones. Immunohistochemical analysis revealed significant activation of transcription factors in diseased gingival tissues. CONCLUSION: The findings of the present study reveal the role of transcription factors NF-κB and STAT3 in periodontal pathogenesis and disease susceptibility of fibroblast subpopulations in periodontal disease could be mediated through activation of NF-κB and STAT3. Since genetic factors are nonmodifyable, transcription factors are promising targets for future host modulation therapy.

Nanosecond laser ablation enhances cellular infiltration in a hybrid tissue scaffold.

Hybrid tissue engineered (HTE) scaffolds constituting polymeric nanofibers and biological tissues have attractive bio-mechanical properties. However, they suffer from small pore size due to dense overlapping nanofibers resulting in poor cellular infiltration. In this study, using nanosecond (ns) laser, we fabricated micro-scale features on Polycaprolactone (PCL)-Chitosan (CH) nanofiber layered bovine pericardium based Bio-Hybrid scaffold to achieve enhanced cellular adhesion and infiltration. The laser energy parameters such as fluence of 25J/cm2, 0.1mm instep and 15 mark time were optimized to get structured microchannels on the Bio-Hybrid scaffolds. Laser irradiation time of 40µs along with these parameters resulted in microchannel width of ~50µm and spacing of ~35µm between adjacent lines. The biochemical, thermal, hydrophilic and uniaxial mechanical properties of the Bio-Hybrid scaffolds remained comparable after laser ablation reflecting extracellular matrix (ECM) stability. Human umbilical cord mesenchymal stem cells and mouse cardiac fibroblasts seeded on these laser-ablated Bio-Hybrid scaffolds exhibited biocompatibility and increased cellular adhesion in microchannels when compared to non-ablated Bio-Hybrid scaffolds. These findings suggest the feasibility to selectively ablate polymer layer in the HTE scaffolds without affecting their bio-mechanical properties and also describe a new approach to enhance cellular infiltration in the HTE scaffolds.

Drug loaded microbeads entrapped electrospun mat for wound dressing application.

A new design of antibiotic loaded wound dressing and its initial in vitro evaluation is described. Chitosan microbeads loaded with ampicillin were sandwiched within polycaprolactone electrospun mat (MbAPPCL). The morphology was analyzed by scanning electron microscopy and surface chemistry was characterized by Fourier Transform Infrared Spectroscopy. In vitro cytotoxicity using L-929 fibroblast cells by direct contact test and elution assay revealed non-cytotoxic nature of MbAPPCL. The cell adhesion and viability analysis further confirmed the cytocompatibility of MbAPPCL as a wound dressing material. Percentage hemolysis and platelet adhesion on the mat exposed to blood substantiated the hemocompatibility. The antibiotic susceptibility test analyzed on Staphylococcus aureus by agar plate method confirmed the drug release and antimicrobial property. The proposed wound dressing model explained with ampicillin as a candidate drug has the potential to include microbeads with different antibiotics for multi drug treatment.

Biological and mechanical evaluation of a Bio-Hybrid scaffold for autologous valve tissue engineering.

Major challenge in heart valve tissue engineering for paediatric patients is the development of an autologous valve with regenerative capacity. Hybrid tissue engineering approach is recently gaining popularity to design scaffolds with desired biological and mechanical properties that can remodel post implantation. In this study, we fabricated aligned nanofibrous Bio-Hybrid scaffold made of decellularized bovine pericardium: polycaprolactone-chitosan with optimized polymer thickness to yield the desired biological and mechanical properties. CD44+, αSMA+, Vimentin+ and CD105- human valve interstitial cells were isolated and seeded on these Bio-Hybrid scaffolds. Subsequent biological evaluation revealed interstitial cell proliferation with dense extra cellular matrix deposition that indicated the viability for growth and proliferation of seeded cells on the scaffolds. Uniaxial mechanical tests along axial direction showed that the Bio-Hybrid scaffolds has at least 20 times the strength of the native valves and its stiffness is nearly 3 times more than that of native valves. Biaxial and uniaxial mechanical studies on valve interstitial cells cultured Bio-Hybrid scaffolds revealed that the response along the axial and circumferential direction was different, similar to native valves. Overall, our findings suggest that Bio-Hybrid scaffold is a promising material for future development of regenerative heart valve constructs in children.

An ex vivo evaluation of the efficacy of andrographolide in modulating differential expression of transcription factors and target genes in periodontal cells and its potential role in treating periodontal diseases.

ETHANOPHARMACOLOGICAL RELEVANCE: Andrographolide is a herbal extract traditionally used in South Asian countries for treating inflammatory diseases. AIM OF THE STUDY: To evaluate the efficacy of andrographolide in management of periodontal disease which is a highly prevalent oral disease. MATERIALS AND METHODS: Periodontal ligament fibroblasts (PDLF) were cultured from healthy and diseased periodontium using explant culture methods. The safe dose of AG was determined using MTT assay. LPS (lipopolysaccharide) of the most important periodontopathogen, P gingivalis was used to activate NF-κB and STAT3 in PDLF. The efficacy of AG in inhibiting NF-κB and STAT3 was analyzed using immunofluorescence. Down regulation of expression of target genes of these transcription factors related to inflammation and bone resorption were analyzed using real time PCR. RESULTS: AG up to the concentration of 25µM was found to be safe as determined by MTT assay. Statistically significant activation of NF-κB and STAT3 in cultured PDLF was observed in diseased group compared to healthy controls before and after LPS challenge. 5µM AG pretreatment significantly inhibited activation of NF-κB and STAT3 and down regulated expression of inflammatory and bone resorptive genes in cultured PDLF. CONCLUSIONS: The findings of the present study propose the adjunctive use of a novel herbal drug andrographolide as a promising host modulation agent for periodontal therapy by inhibiting NF-κB and STAT3 activation and inhibition of inflammation and bone resorption related genes.

Electrospun Cytocompatible Polycaprolactone Blend Composite with Enhanced Wettability for Bone Tissue Engineering.

Electrospinning is recently used in tissue engineering due to their excellent ability to mimic the structure of extra cellular matrix (ECM), a prerequisite for creating an optimal microenvironment for cell growth. Electrospun nanofibrous composite scaffolds consisting of polycaprolactone (PCL)/Poly(1,4-butylene adipate-co-polycaprolactam) (PBAPCL) blend with hydroxyapatite (HA) have been fabricated to enhance the wettability and osseointegrative properties. Fourier transform-infrared spectroscopy (FT-IR) confirmed molecular interactions of the polymer blend along with the presence of HA. X-ray diffraction analysis (XRD) indicated semi-crystalline nature of the mat and also the presence of HA in the composite mat. The morphology of the fibres, were analyzed using scanning electron microscopy (SEM) and the diameter was found to be in the range of 400­600 nm. The composite fibers were of larger diameter compared to their polymer counterparts. Improved wettability of the electrospun composite mat has been observed by contact angle analysis. In vitro cell culture studies by Live/Dead assay and MTT using human osteosarcoma (HOS) cells indicated the cytocompatible nature of electrospun mat which was further confirmed by cell adhesion using SEM and Actin-phalloidin staining. Addition of PBAPCL and HA to PCL have a beneficial effect on cell growth and proliferation thereby making the composite, a prospective scaffold for bone tissue engineering applications.

Microgravity as a means to incorporate HepG2 aggregates in polysaccharide-protein hybrid scaffold.

Tissue culture under microgravity provides a venue which promotes cell-cell association while avoiding the detrimental effects of high shear stress. Hepatocytes cultured on carriers or entrapped within matrices under simulated microgravity conditions showed improved cell function and proliferation. In the present study, a new approach was adopted where a non-cell adherent scaffold was incorporated with hepatospheroids (HepG2) under microgravity. Gum arabic (GA) was cross-linked with gelatin (GA-Gel) and collagen (GA-Col) to prepare non-cell adherent scaffolds. Microgravity experiments with GA-Gel and GA-Col indicated that GA-Col is a better substrate compared to GA-Gel. Microgravity experiments of GA-Col scaffolds with HepG2 cells confirmed that the non-adherent surface with porous architecture can incorporate hepatocyte spheroids and maintain liver specific functions. Albumin and urea synthesis of hepatocytes was sustained up to 6 days under microgravity conditions in the presence of GA-Col scaffold. This new approach of using non-cell adherent matrix and microgravity environment for developing biological substitutes will be beneficial in tissue engineering, bioartificial liver devices and in vitro safety assessment of drugs.

Gum arabic-curcumin conjugate micelles with enhanced loading for curcumin delivery to hepatocarcinoma cells.

Curcumin is conjugated to gum arabic, a highly water soluble polysaccharide to enhance the solubility and stability of curcumin. Conjugation of curcumin to gum arabic is confirmed by (1)H NMR, fluorescence and UV spectroscopy studies. The conjugate self assembles to spherical nano-micelles (270 ± 5 nm) spontaneously, when dispersed in aqueous medium. Spherical morphology of the self assembled conjugate is evidenced by field emission scanning electron microscopy and transmission electron microscopy. The self assembly of the amphiphilic conjugate into micelle in aqueous medium significantly enhances the solubility (900 fold of that of free curcumin) and stability of curcumin in physiological pH. The anticancer activity of the conjugate micelles is found to be higher in human hepatocellular carcinoma (HepG2) cells than in human breast carcinoma (MCF-7) cells. The conjugate exhibits enhanced accumulation and toxicity in HepG2 cells due to the targeting efficiency of the galactose groups present in gum arabic.

Engineering of a polymer layered bio-hybrid heart valve scaffold.

Current treatment strategy for end stage valve disease involves either valvular repair or replacement with homograft/mechanical/bioprosthetic valves. In cases of recurrent stenosis/ regurgitation, valve replacement is preferred choice of treatment over valvular repair. Currently available mechanical valves primarily provide durability whereas bioprosthetic valves have superior tissue compatibility but both lack remodelling and regenerative properties making their utility limited in paediatric patients. With advances in tissue engineering, attempts have been made to fabricate valves with regenerative potential using various polymers, decellularized tissues and hybrid scaffolds. To engineer an ideal heart valve, decellularized bovine pericardium extracellular matrix (DBPECM) is an attractive biocompatible scaffold but has weak mechanical properties and rapid degradation. However, DBPECM can be modified with synthetic polymers to enhance its mechanical properties. In this study, we developed a Bio-Hybrid scaffold with non-cross linked DBPECM in its native structure coated with a layer of Polycaprolactone-Chitosan (PCL-CH) nanofibers that displayed superior mechanical properties. Surface and functional studies demonstrated integration of PCL-CH to the DBPECM with enhanced bio and hemocompatibility. This engineered Bio-Hybrid scaffold exhibited most of the physical, biochemical and functional properties of the native valve that makes it an ideal scaffold for fabrication of cardiac valve with regenerative potential.

Characterization and in vitro evaluation of electrospun chitosan/polycaprolactone blend fibrous mat for skin tissue engineering.

The electrospinning technique allows engineering biomimetic scaffolds within micro to nanoscale range mimicking natural extracellular matrix (ECM). Chitosan (CS) and polycaprolactone (PCL) were dissolved in a modified solvent mixture consisting of formic acid and acetone (3:7) and mixed in different weight ratios to get chitosan-polycaprolactone [CS-PCL] blend solutions. The CS-PCL blend polymer was electrospun in the same solvent system and compared with PCL. The physicochemical characterization of the electrospun fibrous mats was done using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile test, swelling properties, water contact angle (WCA) analysis, surface profilometry and thermo gravimetric analysis (TGA). The CS-PCL fibrous mat showed decreased hydrophobicity. The CS-PCL mats also showed improved swelling property, tensile strength, thermal stability and surface roughness. The cytocompatibility of the CS-PCL and PCL fibrous mats were examined using mouse fibroblast (L-929) cell line by direct contact and cellular activity with extract of materials confirmed non-cytotoxic nature. The potential of CS-PCL and PCL fibrous mats as skin tissue engineering scaffolds were assessed by cell adhesion, viability, proliferation and actin distribution using human keratinocytes (HaCaT) and L-929 cell lines. Results indicate that CS-PCL is a better scaffold for attachment and proliferation of keratinocytes and is a potential material for skin tissue engineering.

Wound healing potential of scaffolds prepared from porcine jejunum and urinary bladder by a non-detergent/enzymatic method.

Scaffolds prepared using extracellular matrices of mammalian organs/tissues, when used as grafts, have wound healing potential. This paper evaluated the physical properties and in vivo wound healing potential of jejunum-derived scaffold (JDS) and urinary bladder-derived scaffold (UDS) of porcine origin prepared by a non-detergent/enzymatic method. The former had higher flexural rigidity and suture retention strength compared to the latter, but both of them had the essential flexural rigidity and suture retention strength required for skin grafts. Full thickness skin-wounds on rabbit dorsum were treated with these scaffolds and the wound healing ability was compared by studying histomorphology parameters such as re-epithelialisation, collagen deposition, angiogenesis, proliferation of cells, mesenchymal cell infiltration and myofibroblast response. The extent of these reactions was assessed using histomorphometry. The results indicated that both grafts initiated healing faster than those wounds without any graft, as evidenced by the extent of cell proliferation and mesenchymal cell infiltration. The myofibroblast response persisted longer in the non-graft assisted wound healing reaction compared to the healing in the graft assisted wounds. Moreover, the JDS induced higher cell proliferation and greater angiogenesis than UDS probably indicating better healing by the former. The results suggested that JDS and UDS prepared by non-detergent/enzymatic method have potential clinical applications.

Liver tissue engineering and cell sources: issues and challenges.

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.

Effect of chronic inflammation and immune response on regeneration induced by decellularized bovine pericardium.

Decellularised tissue produces a variety of host responses ranging from constructive remodeling to scarring on account of its differences in the source of tissue, processing or sterilization methods. In this study, in vivo regeneration induced by decellularised bovine pericardium with or without mild glutaraldehyde crosslinking was studied in relation to its immune and inflammatory response using rat abdominal regeneration model. Mild glutaraldehyde crosslinking was done to subdue inflammatory and immune response without compromising host cell incorporation and graft remodeling. Evaluations were done at both 21 and 90 days. Un-crosslinked decellularised bovine pericardium showed more intense macrophage response predominantly of M2 phenotype at 90 days indicating chronic inflammatory response compared to mildly crosslinked group. This group also showed significant increase in plasma cell and lymphocyte count indicating immune stimulation. Lymphocyte transformation test detected presence of bovine pericardial antigen sensitized lymphocytes at both periods in un-crosslinked group. Lymphocytes from mildly crosslinked group failed to respond in this test at both periods. Significantly higher antibody response was also noted at both periods in un-crosslinked group. However, abdominal wall regeneration was observed only in animals implanted with un-crosslinked decellularised bovine pericardium at 90 days. From the above findings, it is inferred that un-crosslinked decellularised bovine pericardium produced significant chronic inflammatory response at 90 days and stimulated both humoral and cell mediated immune response in comparison to mildly crosslinked decellularised bovine pericardium. Yet this group produced skeletal muscle formation within graft at 90 days.