Small extracellular vesicle-loaded bevacizumab reduces the frequency of intravitreal injection required for diabetic retinopathy.

Diabetic retinopathy (DR) is associated with retinal neovascularization, hard exudates, inflammation, oxidative stress and cell death, leading to vision loss. Anti-vascular endothelial growth factor (Anti-VEGF) therapy through repeated intravitreal injections is an established treatment for reducing VEGF levels in the retina for inhibiting neovascularization and leakage of hard exudates to prevent vision loss. Although anti-VEGF therapy has several clinical benefits, its monthly injection potentially causes devastating ocular complications, including trauma, intraocular hemorrhage, retinal detachment, endophthalmitis, etc. Methods: As mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) demonstrated safety in clinical studies, we have tested the efficacy of MSC-derived small EVs (MSC-sEVs) loaded anti-VEGF drug bevacizumab in a rat model of DR. Results: The study identified a clinically significant finding that sEV loaded with bevacizumab reduces the frequency of intravitreal injection required for treating diabetic retinopathy. The sustained effect is observed from the reduced levels of VEGF, exudates and leukostasis for more than two months following intravitreal injection of sEV loaded with bevacizumab, while bevacizumab alone could maintain reduced levels for about one month. Furthermore, retinal cell death was consistently lower in this period than only bevacizumab. Conclusion: This study provided significant evidence for the prolonged benefits of sEVs as a drug delivery system. Also, EV-mediated drug delivery systems could be considered for clinical application of retinal diseases as they maintain vitreous clarity in the light path due to their composition being similar to cells.

Polyphenol-based targeted therapy for oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic, progressive, and precancerous condition mainly caused by chewing areca nut. Currently, OSF therapy includes intralesional injection of corticosteroids with limited therapeutic success in disease management. Therefore, a combined approach of in silico, in vitro and in vivo drug development can be helpful. Polyphenols are relatively safer than other synthetic counterparts. We used selected polyphenols to shortlist the most suitable compound by in silico tools. Based on the in silico results, epigallocatechin-3-gallate (EGCG), quercetin (QUR), resveratrol, and curcumin had higher affinity and stability with the selected protein targets, transforming growth factor beta-1 (TGF-ß1), and lysyl oxidase (LOX). The efficacy of selected polyphenols was studied in primary buccal mucosal fibroblasts followed by in vivo areca nut extract induced rat OSF model. In in vitro studies, the induced fibroblast cells were treated with EGCG and QUR. EGCG was safer at higher concentrations and more efficient in reducing TGF-ß1, collagen type-1A2 and type-3A1 mRNA expression than QUR. In vivo studies confirmed that the EGCG hydrogel was efficient in improving the disease conditions compared to the standard treatment betamethasone injection with significant reduction in TGF-ß1 and collagen concentrations with increase in mouth opening. EGCG can be considered as a potential, safer and efficient phytomolecule for OSF therapy and its mucoadhesive topical formulation help in the improvement of patient compliance without any side effects. Highlights Potential polyphenols were shortlisted to treat oral submucous fibrosis (OSF) using in silico tools Epigallocatechin 3-gallate (EGCG) significantly reduced TGF-ß1 and collagen both in vitro and in vivo EGCG hydrogel enhanced antioxidant defense, modulated inflammation by reducing TGF-ß1 and improved mouth opening in OSF rat model.

An overview of the current advances in the treatment of inflammatory diseases using mesenchymal stromal cell secretome.

The growing interest in mesenchymal stromal cell (MSC) therapy has been leading to the utilization of its therapeutic properties in a variety of inflammatory diseases. The clinical translation of the related research from bench to bedside is cumbersome due to some obvious limitations of cell therapy. It is evident from the literature that the MSC secretome components mediate their wide range of functions. Cell-free therapy using MSC secretome is being considered as an emerging and promising area of biotherapeutics. The secretome mainly consists of bioactive factors, free nucleic acids, and extracellular vesicles. Constituents of the secretome are greatly influenced by the cell's microenvironment. The broad array of immunomodulatory properties of MSCs are now being employed to target inflammatory diseases. This review focuses on the emerging MSC secretome therapies for various inflammatory diseases. The mechanism of action of the various anti-inflammatory factors is discussed. The potential of MSC secretome as a viable anti-inflammatory therapy is deliberated.

Matrilin3/TGFß3 gelatin microparticles promote chondrogenesis, prevent hypertrophy, and induce paracrine release in MSC spheroid for disc regeneration.

Degenerative disc disease (DDD) is the leading cause of excruciating lower back pain and disability in adults worldwide. Among the current treatments for DDD, cell-based therapies such as the injection of both disc- and non-disc-derived chondrocytes have shown significant improvements in the patients' condition. However, further advancement of these therapies is required to not only ensure a supply of healthy chondrocytes but also to promote regeneration of the defective cells in the injury site. Here, we report that the incorporation of gelatin microparticles coloaded with transforming growth factor beta 3 and matrilin 3 promoted chondrogenic differentiation of adipose-derived mesenchymal stem cell spheroids while preventing hypertrophy and terminal differentiation of cells. Moreover, these composite spheroids induced the release of chondrogenic cytokines that, in turn, promoted regeneration of degenerative chondrocytes in vitro. Finally, injections of these composite spheroids in a rat model of intervertebral disc disease promoted restoration of the chondrogenic properties of the cells, thereby allowing regeneration of the chondrogenic tissue in vivo.

Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-ß, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-ß2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.

Efficacy of matrilin-3-primed adipose-derived mesenchymal stem cell spheroids in a rabbit model of disc degeneration.

BACKGROUND: Chronic low back pain is a prevalent disability, often caused by intervertebral disc (IVD) degeneration. Mesenchymal stem cell (MSC) therapy could be a safe and feasible option for repairing the degenerated disc. However, for successful translation to the clinic, various challenges need to be overcome including unwanted adverse effects due to acidic pH, hypoxia, and limited nutrition. Matrilin-3 is an essential extracellular matrix (ECM) component during cartilage development and ossification and exerts chondrocyte protective effects. METHODS: This study evaluated the effects of matrilin-3-primed adipose-derived MSCs (Ad-MSCs) on the repair of the degenerated disc in vitro and in vivo. We determined the optimal priming concentration and duration and developed an optimal protocol for Ad-MSC spheroid generation. RESULTS: Priming with 10 ng/ml matrilin-3 for 5 days resulted in the highest mRNA expression of type 2 collagen and aggrecan in vitro. Furthermore, Ad-MSC spheroids with a density of 250 cells/microwell showed the increased secretion of favorable growth factors such as transforming growth factor beta (TGF-ß1), TGF-ß2, interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and matrix metalloproteinase 1 (MMP1) and decreased secretion of hypertrophic ECM components. In addition, matrilin-3-primed Ad-MSC spheroid implantation was associated with optimal repair in a rabbit model. CONCLUSION: Our results suggest that priming MSCs with matrilin-3 and spheroid formation could be an effective strategy to overcome the challenges associated with the use of MSCs for the treatment of IVD degeneration.

A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome.

BACKGROUND: Mesenchymal stromal cell conditioned medium (MSC-CM) contains a cocktail of bioactive factors that act synergistically to induce therapeutic effects. This has been clearly demonstrated by in vivo applications of MSC-CM, but the establishment of controlled delivery systems is an unmet requirement for clinical translation. METHODS: We developed a nanocomposite-hydrogel (NP-H) comprised of poly-L-lactide nanoparticles (NPs) embedded in gelatin/hyaluronic acid (Gel/HA) hydrogel as a delivery vehicle for MSC-CM. First, we optimized the culture conditions for bone marrow-derived MSCs using serum-containing medium (SCM) and serum-free medium (SFM) and characterized the corresponding CM (serum-containing conditioned medium (ScCM) and serum-free conditioned medium (SfCM), respectively) for its potency and xeno markers. Then we prepared a composite matrix followed by physiochemical characterization and functional assays were performed. RESULTS: Nanocomposite hydrogel displayed an even distribution of NPs along with high porosity (> 60%) and swelling ratios > 1500%, while its protein release pattern corresponded to a mix of degradation and diffusion kinetics. Functional evaluation of the composites was determined using MSCs and human fibroblasts (HFFs). The cells seeded directly onto the composites displayed increasing metabolic activities over time, with ScCM-NP-H groups having maximum activity. The cells treated in vitro with 5% and 10% extracts of ScCM-NP-H and SfCM-NP-H exhibited a dose- and duration-dependent response. Cell activities reduced considerably for all groups, except 10% ScCM-NP-H, which displayed a significant increase over time. CONCLUSION: We observed that sustained release of MSC-CM is required to prevent dose-dependent cytotoxicity. The proposed nanocomposite hydrogel for MSC-CM delivery can open up a new array for its clinical application.

Matrix Metalloproteinase-8 Inhibition Prevents Disruption of Blood-Spinal Cord Barrier and Attenuates Inflammation in Rat Model of Spinal Cord Injury.

After spinal cord injury (SCI), tight junction (TJ) protein degradation increases permeability and disrupts the blood-spinal cord barrier (BSCB). The BSCB is primarily formed of endothelial cell, which forms a specialized tight seal due to the presence of TJs. BSCB disruption after SCI allows neutrophil infiltration. Matrix metalloproteinase (MMP)-8 is believed to be mainly expressed by neutrophils and is quickly released upon neutrophil activation. Here, we determined whether MMP-8 is involved in the TJ protein degradation in endothelial cells and also determined its role in the neuroinflammation after SCI. MMP-8 recombinant protein treatment increases the TNF-α expression and decreased the TJ (occludin and zonula occludens-1) protein expression in the endothelial cells. Likewise, specific MMP-8 inhibitor (MMP-8I) significantly prevented the TNF-α-induced decrease in the expression of TJ protein in endothelial cells. Furthermore, MMP-8 expression was significantly increased 1 and 3 days after moderate compression (35 g for 5 min at T10 level) SCI, whereas TJ protein levels decreased as determined qRT-PCR, western blotting, and immunohistochemistry. MMP-8 was inhibited directly using a MMP-8I (5 mg/kg) and indirectly by reducing neutrophil infiltration with sivelestat sodium (50 mg/kg) or using the antioxidant N-acetyl-L-cysteine (100 mg/kg). The MMP-8I significantly decreased TNF-α expression, IL-6, and iNOS expression and increased TJ protein expression after SCI. In addition, MMP-8I significantly lessens the amount of Evans blue dye extravasation observed after injury. Thus, our result suggests that MMP-8 plays an imperative role in inflammation and degradation of TJ proteins. Increased MMP-8 expression was associated with the early inflammatory phase of SCI. Inhibiting MMP-8 significantly attenuated SCI-induced inflammation, BSCB breakdown, and cell injury.

Matrilin-3 codelivery with adipose-derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model.

Matrilin-3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin-3 exerts chondroprotective effects by regulating an anti-inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin-3 with infrapatellar adipose-tissue-derived mesenchymal stem cells (Ad-MSCs) may enhance articular cartilage regeneration. Matrilin-3 treatment of Ad-MSCs in serum-free media induced collagen II and aggrecan expression, and matrilin-3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin-3 codelivery with Ad-MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad-MSCs and hyaluronic acid were implanted at the defect site with or without matrilin-3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin-3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140-ng matrilin-3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin-3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140-ng matrilin-3 revealed significant hyaline-like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700-ng matrilin-3 exhibited drastically reduced cartilage regeneration with mixed hyaline-fibrocartilage morphology. Codelivery of matrilin-3 with Ad-MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue-specific protein for a cartilage-targeted stem cell therapy.

Matrilin-3 Role in Cartilage Development and Osteoarthritis.

The extracellular matrix (ECM) of cartilage performs essential functions in differentiation and chondroprogenitor cell maintenance during development and regeneration. Here, we discuss the vital role of matrilin-3, an ECM protein involved in cartilage development and potential osteoarthritis pathomechanisms. As an adaptor protein, matrilin-3 binds to collagen IX to form a filamentous network around cells. Matrilin-3 is an essential component during cartilage development and ossification. In addition, it interacts directly or indirectly with transforming growth factor ß (TGF-ß), and bone morphogenetic protein 2 (BMP2) eventually regulates chondrocyte proliferation and hypertrophic differentiation. Interestingly, matrilin-3 increases interleukin receptor antagonists (IL-Ra) in chondrocytes, suggesting its role in the suppression of IL-1ß-mediated inflammatory action. Matrilin-3 downregulates the expression of matrix-degrading enzymes, such as a disintegrin metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ADAMTS5, matrix metalloproteinase 13 (MMP13), and collagen X, a hypertrophy marker during development and inflammatory conditions. Matrilin-3 essentially enhances collagen II and aggrecan expression, which are required to maintain the tensile strength and elasticity of cartilage, respectively. Interestingly, despite these attributes, matrilin-3 induces osteoarthritis-associated markers in chondrocytes in a concentration-dependent manner. Existing data provide insights into the critical role of matrilin-3 in inflammation, matrix degradation, and matrix formation in cartilage development and osteoarthritis.

Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

Serum paraoxonase 1 activity status in patients with liver disorders.

BACKGROUND/AIM: Paraoxonase 1 (PON1) is an esterase, exclusively synthesized by liver. The present study has two objectives: to determine the PON1 activity status in various disorders associated with hepatocellular damage and to correlate the changes of PON1 activity with the standard liver function and fasting lipid profile tests in these disorders. PATIENTS AND METHODS: The study groups consisted of 95 patients with liver diseases including acute viral hepatitis (14), cirrhosis with portal hypertension (33), leptospirosis (14), sepsis and multi organ failure (15), left ventricular failure (9), and falciparum malaria (10); and 53 healthy controls. Serum PON1 activity was measured manually using spectrophotometer. Liver function test parameters and fasting lipid profile were performed in clinical chemistry auto analyzer (Hitachi 912). RESULTS: The serum PON1 activity in patients with acute viral hepatitis and sepsis decreased significantly ( P < 0.001) and moderately in falciparum malaria ( P < 0.05). However, in patients with cirrhosis, leptospirosis and left ventricular patients, its activity did not change significantly. On applying Pearson correlation, serum PON1 activity correlated positively with high-density lipoprotein-cholesterol (HDL-C) in patients with sepsis (r=0.633, P < 0.05), left ventricular failure patients (r=0.814, P < 0.05) and negatively with acute viral hepatitis patients (r=-0.528, P <0.05). CONCLUSION: PON1 activity has decreased significantly in acute viral hepatitis, sepsis with multi organ failure and falciparum malaria patients. Determination of PON1 activity may serve as a useful additional test in assessing these conditions.

Copper and ceruloplasmin levels in relation to total thiols and GST in type 2 diabetes mellitus patients.

Presence of oxidative stress in type 2 diabetes mellitus (DM) is well proved. Current study was undertaken to know the relation between fasting plasma glucose (FPG) and copper along with antioxidants like total thiols and ceruloplasmin, and antioxidant enzyme glutathione S transferase (GST). The study group consisted of a total of 201 subjects which included nondiabetic healthy control subjects (n = 78) and diabetic patients (n = 123). Plasma total thiols, GST, copper and ceruloplasmin levels were measured all the subjects using spectrophotometric methods and FPG levels were determined in clinical chemistry analyzer Hitachi 912. There was significant increase in FPG (P<0.001) and copper (P<0.001) and decrease in ceruloplasmin (P<0.001) and protein thiols (P<0.001) in type 2 DM cases compared to healthy controls. There was no significant change in GST between type 2 DM cases and controls. There was significant negative correlation of FPG with antioxidants like ceruloplasmin (r = -0.420, P<0.001) and total thiols (r = -0.565, P<0.001). Protein thiols correlated positively with ceruloplasmin (r = 0.364, P<0.001). Our study indicates possible increase in copper mediated generation of ROS leading to increased consumption of available antioxidants in the body.

Petroleum ether extract of Cissus quadrangularis (Linn.) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis.

OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. METHODS: MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 microg/mL) were also subjected to a cell proliferation assay (MTT). RESULTS: Treatment with 100, 200 or 300 microg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 microg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. CONCLUSION: The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/ alternative natural medicine for bone diseases such as osteoporosis.

Sporadic Creutzfeldt-Jakob disease--a review.

The objective is to study a patient with sporadic Creutzfeldt-Jakob disease (CJD). The patient, a 70-year-old woman with a history spanning over 1 month, with acute onset, progressive abnormal behavior, and cognitive decline with generalized asymmetrical myoclonic jerking, startle phenomenon, and cortical blindness, was referred to the hospital. On observation of clinical symptoms, metabolic and hematological investigations, MRI (magnetic resonance imaging), and EEG (electroencephalogram) were done. The clinical symptoms, MRI, and diagnostic EEG were suggestive of sporadic CJD. Other metabolic encephalopathies were ruled out. With sodium valproate and clonezepam, her myoclonic jerks improved slightly. As CJD is an incurable disease, no definitive treatment could be given.

Petroleum ether extract of Cissus quadrangularis (Linn. ) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis

OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. METHODS: MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 µg/mL) were also subjected to a cell proliferation assay (MTT). RESULTS: Treatment with 100, 200 or 300 µg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 µg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. CONCLUSION: The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/alternative natural medicine for bone diseases such as osteoporosis.