Shiplu Roy Chowdhury

Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing

BACKGROUND AIMS Platelet-rich plasma (PRP) has been found to contain a high concentration of growth factors that are present during the process of healing. Studies conducted found that application of PRP accelerates wound healing. In this study, we characterized the skin cell suspension harvested using the co-isolation technique and evaluated the effects of PRP (10% and 20%, v/v) on co-cultured keratinocytes and fibroblasts in terms of wound healing. METHODS Human keratinocytes and fibroblasts were harvested via co-isolation technique and separated via differential trypsinization. These cells were then indirectly co-cultured in medium supplemented with 10% or 20% PRP for 3 days without medium change for analysis of wound-healing potential. The wound-healing potential of keratinocytes and fibroblasts was evaluated in terms of growth property, migratory property, extracellular matrix gene expression and soluble factor secretion. RESULTS The co-isolation technique yielded a skin cell population dominated by fibroblasts and keratinocytes, with a small amount of melanocytes. Comparison between the 10% and 20% PRP cultures showed that the 10% PRP culture exhibited higher keratinocyte apparent specific growth rate, and secretion of hepatocyte growth factor, monocyte chemoattractant protein-1, epithelial-derived neutrophil-activating protein 78 and vascular endothelial growth factor A, whereas the 20% PRP culture has significantly higher collagen type 1 and collagen type 3 expressions and produced more granulocyte-macrophage colony-stimulating factor. CONCLUSIONS PRP concentration modulates keratinocyte and fibroblast wound healing potential, whereby the 10% PRP promotes wound remodeling, whereas the 20% PRP enhances inflammation and collagen deposition.

Aqueous extract of Centella asiatica promotes corneal epithelium wound healing in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Centella asiatica is a traditional herbal medicine that has been shown to have pharmacological effect on skin wound healing, and could be potential therapeutic agent for corneal epithelial wound healing. AIM OF THE STUDY This study was done to evaluate the effects of Centella asiatica on the proliferation and migration of rabbit corneal epithelial (RCE) cells in the in vitro wound healing model. MATERIALS AND METHODS RCE cells were cultured with or without supplementation of Centella asiatica aqueous extract. Viability and proliferation of the RCE cells was determined by MTT assay and cell cycle was analyzed by flow cytometry. In vitro re-epithelization was studied by scratch assay and migration rate was evaluated quantitatively by image analyzer. Expression of corneal specific differentiation markers, CK12 and connexin 43, were studied via RT-PCR. RESULTS It was found that supplementation of Centella asiatica did not show any significant effect on the RCE cells proliferation at the concentration up to 500ppm, while at the concentration of 1000ppm significantly inhibited RCE cells proliferation (p<0.05). However, at the concentration up to 62.5ppm, RCE cells shows significant enhancement of migration rate compared to the control group (p<0.05). It was also found that the supplementation of Centella asiatica aqueous extract did not alter the expression of differentiation markers and cell cycle. CONCLUSION In conclusion, supplementation of Centella asiatica aqueous extract at low concentrations could be useful to promote corneal epithelium wound healing.

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats. Methods RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies. Results No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells. Conclusions Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.

Surface modification of electrospun poly(methyl methacrylate) (PMMA) nanofibers for the development of in vitro respiratory epithelium model.

Scaffold design is an important aspect of in vitro model development. In this study, nanoscaffold surface modification, namely UV radiation and genipin cross-linking to immobilize collagen on the surface of electrospun poly (methyl methacrylate) (PMMA) nanofiber sheet was investigated. Samples were divided into four groups; PMMA nanofibers (PMMA), collagen-coated PMMA nanofibers (PMMACOL), genipin cross-linked collagen-coated PMMA nanofibers (PMMAGEN), and UV-irradiated collagen-coated PMMA nanofibers (PMMAUV). 6 h of UV radiation significantly reduced the hydrophobicity of PMMA nanofibers from (131.88° ± 1.33°) to (110.04° ± 0.27°) (p < 0.05). The amount of collagen immobilized was significantly higher in PMMAGEN group (239.36 ± 16.63 μg collagen/mg nanofibers) (p < 0.05) compared to the other groups. RECs on all scaffold expressed epithelial cell-specific markers (CK18 and CK14), mucin-producing cell marker (MUC5Ac) and were actively proliferating, based on the positive expression of Ki67. Total number of attached cells was significantly the highest in PMMAUV group on day 9 (6.44 × 104 ± 2.77 × 104 cells/cm2) and it has the highest proliferation rate from day 4 to 9 (0.005 ± 0.003 h−1) compared to the other groups. Even though PMMAGEN group showed the highest collagen adsorption, in terms of cells attachment and proliferation, PMMAUV group showed a better outcome compared to the other groups. Thus, PMMAUV scaffold is more suitable to be used in the construction of in vitro respiratory epithelial model.

Cardiomyogenic differentiation of human sternal bone marrow mesenchymal stem cells using a combination of basic fibroblast growth factor and hydrocortisone

The alarming rate of increase in myocardial infarction and marginal success in efforts to regenerate the damaged myocardium through conventional treatments creates an exceptional avenue for cell‐based therapy. Adult bone marrow mesenchymal stem cells (MSCs) can be differentiated into cardiomyocytes, by treatment with 5‐azacytidine, thus, have been anticipated as a therapeutic tool for myocardial infarction treatment. In this study, we investigated the ability of basic fibroblastic growth factor (bFGF) and hydrocortisone as a combined treatment to stimulate the differentiation of MSCs into cardiomyocytes. MSCs were isolated from sternal marrow of patients undergoing heart surgery (CABG). The isolated cells were initially monitored for the growth pattern, followed by characterization using ISCT recommendations. Cells were then differentiated using a combination of bFGF and hydrocortisone and evaluated for the expression of characteristic cardiac markers such as CTnI, CTnC, and Cnx43 at protein level using immunocytochemistry and flow cytometry, and CTnC and CTnT at mRNA level. The expression levels and pattern of the cardiac markers upon analysis with ICC and qRT‐PCR were similar to that of 5‐azacytidine induced cells and cultured primary human cardiomyocytes. However, flow cytometric evaluation revealed that induction with bFGF and hydrocortisone drives MSC differentiation to cardiomyocytes with a marginally higher efficiency. These results indicate that combination treatment of bFGF and hydrocortisone can be used as an alternative induction method for cardiomyogenic differentiation of MSCs for future clinical applications.

Tissue-Engineered Skin Substitute Enhances Wound Healing after Radiation Therapy.

OBJECTIVE: When given in conjunction with surgery for treating cancer, radiation therapy may result in impaired wound healing, which, in turn, could cause skin ulcers. In this study, bilayer and monolayer autologous skin substitutes were used to treat an irradiated wound. MATERIALS AND METHODS: A single dose of 30 Gy of linear electron beam radiation was applied to the hind limb of nude mice before creating the skin lesion (area of 78.6 mm2). Monolayer tissue-engineered skin substitutes (MTESSs) were prepared by entrapping cultured keratinocytes in fibrin matrix, and bilayer tissue-engineered skin substitutes (BTESSs) were prepared by entrapping keratinocytes and fibroblasts in separate layers. Bilayer tissue-engineered skin substitute and MTESS were implanted to the wound area. Gross appearance and wound area were analyzed to evaluate wound healing efficiency. Skin regeneration and morphological appearance were observed via histological and electron microscopy. Protein expressions of transforming growth factor &bgr;1 (TGF-&bgr;1), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor (VEGF) in skin regeneration were evaluated by immunohistochemistry (IHC). RESULTS: Macroscopic observation revealed that at day 13, treatments with BTESS completely healed the irradiated wound, whereas wound sizes of 1.1 ± 0.05 and 6.8 ± 0.14 mm2 were measured in the MTESS-treated and untreated control groups, respectively. Hematoxylin-eosin (H&E) analysis showed formation of compact and organized epidermal and dermal layers in the BTESS-treated group, as compared with MTESS-treated and untreated control groups. Ultrastructural analysis indicates maturation of skin in BTESS-treated wound evidenced by formation of intermediate filament bundles in the dermal layer and low intercellular space in the epidermal layer. Expressions of TGF-&bgr;1, PDGF-BB, and VEGF were also higher in BTESS-treated wounds, compared with MTESS-treated wounds. CONCLUSIONS: These results indicate that BTESS is the preferred treatment for irradiated wound ulcers.

The effects of human serum to the morphology, proliferation and gene expression level of the respiratory epithelium in vitro.

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80-90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p<0.05). However, there were no significant differences in the viability of RE cells in both groups (p=0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p>0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in vitro thus producing a better quality of respiratory epithelium for future tracheal reconstruction.

Identification of suitable culture condition for expansion and osteogenic differentiation of human bone marrow stem cells

This study was undertaken in order to identify the best culture strategy to expand and osteogenic differentiation of human bone marrow stem cells (hBMSCs) for subsequent bone tissue engineering. In this regard, the experiment was designed to evaluate whether it is feasible to bypass the expansion phase during hBMSCs differentiation towards osteogenic lineages by early induction, if not identification of suitable culture media for enhancement of hBMSCs expansion and osteogenic differentiation. It was found that introduction of osteogenic factors in alpha-minimum essential medium (αMEM) during expansion phase resulted in significant reduction of hBMSCs growth rate and osteogenic gene expressions. In an approach to identify suitable culture media, the growth and differentiation potential of hBMSCs were evaluated in αMEM, F12:DMEM (1:1; FD), and FD with growth factors. It was found that αMEM favors the expansion and osteogenic differentiation of hBMSCs compared to that in FD. However, supplementation of growth factors in FD, only during expansion phase, enhances the hBMSCs growth rate and significantly up-regulates the expression of CBFA-1 (the early markers of osteogenic differentiation) during expansion, and, other osteogenic genes at the end of induction compared to the cells in αMEM and FD. These results suggested that the expansion and differentiation phase of the hBMSCs should be separately and carefully timed. For bone tissue engineering, supplementation of growth factors in FD only during the expansion phase was sufficient to promote hBMSCs expansion and differentiation, and preferably the most efficient culture condition.

Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases.