Evaluation of rabbit adipose derived stem cells fate in perfused multilayered silk fibroin composite scaffold for Osteochondral repair.

Development of osteochondral tissue engineering approaches using scaffolds seeded with stem cells in association with mechanical stimulations has been recently considered as a promising technique for the repair of this tissue. In this study, an integrated and biomimetic trilayered silk fibroin (SF) scaffold containing SF nanofibers in each layer was fabricated. The osteogenesis and chondrogenesis of stem cells seeded on the fabricated scaffolds were investigated under a perfusion flow. 3-Dimethylthiazol-2,5-diphenyltetrazolium bromide assay showed that the perfusion flow significantly enhanced cell viability and proliferation. Analysis of gene expression by stem cells revealed that perfusion flow had significantly upregulated the expression of osteogenic and chondrogenic genes in the bone and cartilage layers and downregulated the hypertrophic gene expression in the intermediate layer of the scaffold. In conclusion, applying flow perfusion on the prepared integrated trilayered SF-based scaffold can support osteogenic and chondrogenic differentiation for repairing osteochondral defects.

The effect of light compressive and tensile mechanical forces on SOST and POSTN expressions in human periodontal ligament cells: an in vitro study.

Periodontal ligament (PDL) cells play an important role in mechanosensing and secretion of signaling molecules during bone remodeling. However, the regulatory mechanism is unknown. The aim of the present study is to investigate the expression pattern of periostin and sclerostin in response to orthodontic forces in periodontal ligament cells in vitro. PDL cells were isolated from extracted teeth and treated with compressive forces of 25 gr/cm2 or equiaxial tension forces at frequency 1 Hz for 0, 24, 48, and 72 h. qRT-PCR was applied to evaluate the gene expressions. The secretion of sclerostin and periostin was assessed using ELISA. DAPI staining was used to evaluate apoptosis. The expression of sclerostin elevated significantly at protein and gene levels under compression forces after 24 h, while the application of tensile forces induced the expression of periostin and its upstream regulator RUNX2 (p < 0.05). Gene expression up-regulation was significant for POSTN and RUNX2 after 48 and 72 h tensile forces. Also, the gene expression of sclerostin reduced in a time-dependent manner after application of tensile force. The compression forces enhanced apoptosis to 7.5 ± 3.5% and induced gene expression of apoptotic markers of CASP9, and BCL2 within 72 h of exposure. Periostin and sclerostin play an important role in orthodontic loads and their expressions are affected oppositely by compressive and tensile forces that might be suggested as a biomarker for assessment of bone remodeling during orthodontic treatment.

Biomimetic vascular tissue engineering by decellularized scaffold and concurrent cyclic tensile and shear stresses.

Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at -20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress-strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues.

The Role of Low-Frequency Electromagnetic Fields on Mesenchymal Stem Cells Differentiation: A Systematic Review.

BACKGROUND: Low-frequency electromagnetic fields (EMFs) influence biological processes. This present study was aimed at the scientific literature on the use of EMFs in the mesenchymal stem cell differentiation process. MATERIALS AND METHODS: The electronic search was carried out in PubMed and Web of Science, a database with a combination of the sinusoidal and pulsed low- and extremely low-frequency electromagnetic fields stimulation and mesenchymal stem cells differentiation, considering the period of publication until December 2021. The literature search identified 118 references in PubMed and Web of Science of which 46 articles were selected, respectively, according to the eligibility requirements. CONCLUSION: The analysis of research indicated that EMFs are an easy-to-apply and practical way in cell therapy and tissue engineering when regulation of stem cells is required. Studies have shown that EMFs have positive effects on stem cell differentiation, accelerating its process regardless of the parameters and type of stem cells. However, the exact amplitude, frequency, duration of the electrical field, and application method remain elusive and need more study in future work.

In vitrostatic and dynamic cell culture study of novel bone scaffolds based on 3D-printed PLA and cell-laden alginate hydrogel.

The aim of this paper was to design and fabricate a novel composite scaffold based on the combination of 3D-printed polylactic acid-based triply periodic minimal surfaces (TPMSs) and cell-laden alginate hydrogel. This novel scaffold improves the low mechanical properties of alginate hydrogel and can also provide a scaffold with a suitable pore size, which can be used in bone regeneration applications. In this regard, an implicit function was used to generate some gyroid TPMS scaffolds. Then the fused deposition modeling process was employed to print the scaffolds. Moreover, the micro computed tomography technique was employed to assess the microstructure of 3D-printed TPMS scaffolds and obtain the real geometries of printed scaffolds. The mechanical properties of composite scaffolds were investigated under compression tests experimentally. It was shown that different mechanical behaviors could be obtained for different implicit function parameters. In this research, to assess the mechanical behavior of printed scaffolds in terms of the strain-stress curves on, two approaches were presented: equivalent volume and finite element-based volume. Results of strain-stress curves showed that the finite-element based approach predicts a higher level of stress. Moreover, the biological response of composite scaffolds in terms of cell viability, cell proliferation, and cell attachment was investigated. In this vein, a dynamic cell culture system was designed and fabricated, which improves mass transport through the composite scaffolds and applies mechanical loading to the cells, which helps cell proliferation. Moreover, the results of the novel composite scaffolds were compared to those without alginate, and it was shown that the composite scaffold could create more viability and cell proliferation in both dynamic and static cultures. Also, it was shown that scaffolds in dynamic cell culture have a better biological response than in static culture. In addition, scanning electron microscopy was employed to study the cell adhesion on the composite scaffolds, which showed excellent attachment between the scaffolds and cells.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Replacement of Trypsin by Proteases for Medical Applications.

Background: Cell culture has a crucial role in many applications in biotechnology. The production of vaccines, recombinant proteins, tissue engineering, and stem cell therapy all need cell culture. Most of these activities needed adherent cells to move, which should be trypsinized several times until received on a large scale. Although trypsin is manufactured from the bovine or porcine pancreas, the problem of contamination by unwanted animal proteins, unwanted immune reactions, or contamination to pathogen reagents is the main problem. Objectives: This study investigated microbial proteases as a safe alternative for trypsin replacement in cell culture experiments for the detachment of adherent cells. Methods: The bacteria were isolated from the leather industry effluent based on their protease enzymes. After sequencing their 16S ribosomal deoxyribonucleic acid, their protease enzymes were purified, and their enzyme activities were assayed. The alteration of enzymatic activities using different substrates and the effect of substrate concentrations on enzyme activities were determined. The purified proteases were evaluated for cell detachment in the L929 fibroblast cells compared to trypsin. The separated cells were cultured again, and cell proliferation was determined by the MTT assay. Results: The results showed that the isolated bacteria were Bacillus pumilus, Stenotrophomonas sp., Klebsiella aerogenes, Stenotrophomonas maltophilia, and Bacillus licheniformis. Among the isolated bacteria, the highest and the lowest protease activity belonged to Stenotrophomonas sp. and K. aerogenes, with 60.34 and 11.09 U/mL protease activity, respectively. All the isolated microbial proteases successfully affected L929 fibroblast cells' surface proteins and detached the cells. A significant induction in cell proliferation was observed in the cells treated with K. aerogenes protease and B. pumilus protease, respectively (P < 0.05). Conclusions: The obtained results suggested that microbial proteases can be used as safe and efficient alternatives to trypsin in cell culture in biopharmaceutical applications.

A Microfabrication Method of PCL Scaffolds for Tissue Engineering by Simultaneous Two PDMS Molds Replication.

Not very far away, "tissue engineering" will become one of the most important branches of medical science for curing many types of diseases. This branch needs the cooperation of a wide range of sciences like medicine, chemistry, cellular biology, and genetic and mechanical engineering. Different parameters affect the final produced tissue, but the most important one is the quality and biocompatibility of the scaffold with the desired tissue which can provide the functionality of "native ECM" as well. The quality of the scaffold is directly dependent on its materials, design, and method of fabrication. As to the design and fabrication, there are two main categories: (a) random microporosity such as phase separation, electrospinning, and fused deposition modeling (3D printing) and (b) designed microporosity mostly achievable by stereo lithography and soft lithography. The method of fabrication implemented in this research is a novel method in soft lithography employing a type of "replica molding" with one pair of polydimethylsiloxane (PDMS) molds in contrast to traditional replica molding with just one single mold. In this operation, the solution of polycaprolactone in chloroform is initially prepared, and one droplet of the solution is placed between the molds while a preset pressure is applied to maintain the molds tightly together during the solidification of the polymer layer and vaporization of the solvent. Thus, a perfect warp and woof pattern is created. In this research, it has been approved that this is a feasible method for creating complex patterns and simple straight fiber patterns with different spacings and pore sizes. Cell attachment and migration was studied to find the optimum pore size. It was shown that the small pore size improves the cells' adhesion while reducing cell migration capability within the scaffold.

Erratum to "Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates".

[This corrects the article DOI: 10.1155/2021/5538348.].

Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates.

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

Fabrication and physicochemical characterization of a novel magnetic nanocomposite scaffold: Electromagnetic field effect on biological properties.

In the current research, a novel poly(ε-caprolactone) nanofibrous composite scaffold including CZF-NPs1 (cobalt­zinc ferrite nanoparticles) was investigated to study the physical, mechanical and biological properties of new magnetic nanofibrous materials and then to evaluate the effect of applied electromagnetic field on biological properties of these scaffolds. It was observed that the incorporation of CZF-NPs up to 3 wt.% leads to decrease in nanofibers' diameter to 466 nm. By raising the content of CZF-NPs, hydrophilicity and biodegradation of magnetic nanofibrous scaffolds improved significantly. In addition, the mechanical properties of nanofibers such as stress at break point was interestingly increased in the sample with 3 wt.% of CZF-NPs. The results of biocompatibility, cell adhesion and cell staining assays with L929 cells are much more improved in nanofibers embedded with CZF-NPs in the presence of external electromagnetic field (EMF). According to this study, magnetic nanofibrous scaffolds composed of PCL/CZF-NPs could be considered as a promising candidate to regenerate damaged tissues.

Evaluation of alginate modification effect on cell-matrix interaction, mechanotransduction and chondrogenesis of encapsulated MSCs.

Mesenchymal stem cells (MSCs) are promising cell candidates for cartilage regeneration. Furthermore, it is important to control the cell-matrix interactions that have a direct influence on cell functions. Providing an appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of MSCs. In this study, hydrogels consisted of different proportions of alginates that were modified using gelatin, collagen type I and arginine-glycine-aspartic acid (RGD) and were evaluated regarding their effects on mesenchymal stem cells. The effect of applying hydrostatic pressure on MSCs encapsulated in collagen-modified alginate with and without chondrogenic medium was evaluated 7, 14 and 21 days after culture, which is a comprehensive evaluation of chondrogenesis in 3D hydrogels with mechanical and chemical stimulants. Alcian blue, safranin O and dimethyl methylene blue (DMMB) staining showed the chondrogenic phenotype of cells seeded in the collagen- and RGD-modified alginate hydrogels with the highest intensity after 21 days of culture. The results of real-time PCR for cartilage-specific extracellular matrix genes indicated the chondrogenic differentiation of MSCs in all hydrogels. Also, the synergic effects of chemical and mechanical stimuli are indicated. The highest expression levels of the studied genes were observed in the cells embedded in collagen-modified alginate by loading after 14 days of exposure to the chondrogenic medium. The effect of using IHP on encapsulated MSCs in modified alginate with collagen type I is equal or even higher than using TGF-beta on encapsulated cells. The results of immunohistochemical assessments also confirmed the real-time PCR data.

The chromene derivative 4-Clpgc inhibits cell proliferation and induces apoptosis in the K562 cell line.

Chronic myeloid leukemia (CML) is a malignant blood disease with a particular chromosomal aberration that is known as a common form of leukemia. The chromene family exhibits strong anti-cancer effects. Therefore, the effects of six members of the dihydropyrano [2,3-g] chromene family on cell toxicity and apoptosis induction in K562 cancer cells were investigated and compared with those of normal peripheral blood mononuclear cells (PBMCs). The K562 cells were cultured in the presence of the aforementioned chromene derivatives at concentrations of 40 to 200 µM for 24 to 72 h. The effects of these compounds on the growth and viability of the K562 cell line and PBMCs were studied through MTT assay. Furthermore, apoptosis induction was investigated using flow cytometry. Real-time PCR was used for relative quantification of BCL2, Bax, TP53 and BCR- ABL genes after 48 h of exposing K562 cells and PBMCs to 4-Clpgc. Based on the results, these chromene derivatives inhibited the growth of K562 cells. According to the obtained data, 4-Clpgc was the strongest compound with IC50 values of 102 ± 1.6 µM and 143 ± 9.41 µM in K562 cells and PBMCs, while pgc was the weakest one with IC50 levels of 278 ± 2.7 µM and 366 ± 47 µM in K562 cells and PBMCs (after 72 h), respectively. The results demonstrated that the apoptotic cell percentage in the control group increased from 6.09% to 84.10% and 17.2% to 20.06% in K562 cells and PBMCs after 48 h of treatment, respectively. Moreover, 4-Clpgc treatment increased the expression of Bax and TP53 genes by 42.74 and 35.88 folds in K562 cells and 9.60 and 7.75 folds in PBMCs, respectively. On the other hand, the expression of BCL2 was reduced by 1.47 and 1.38 folds in K562 cells and PBMCs, respectively. These compounds were associated with less toxic effects on normal cells, compared to the cancer cells. In conclusion, these derivatives can be considered as appropriate candidates for leukemia treatment.

Combination of Mechanical and Chemical Methods Improves Gene Delivery in Cell-based HIV Vaccines.

OBJECTIVE: Novel vaccination approaches are required to control human immunodeficiency virus (HIV) infections. The membrane proximal external region (MPER) of Env gp41 subunit and the V3/glycans of Env gp120 subunit were known as potential antigenic targets for anti-HIV-1 vaccines. In this study, we prepared the modified dendritic cells (DCs) and mesenchymal stem cells (MSCs) with HIV-1 MPER-V3 gene using mechanical and chemical approaches. METHODS: At first, MPER-V3 fusion DNA delivery was optimized in dendritic cells (DCs) and mesenchymal stem cells (MSCs) using three mechanical (i.e., uniaxial cyclic stretch, equiaxial cyclic stretch and shear stress bioreactors), and two chemical (i.e., TurboFect or Lipofectamine) methods. Next, the modified DCs and MSCs with MPER-V3 antigen were compared to induce immune responses in vivo. RESULTS: Our data showed that the combination of equiaxial cyclic stretch loading and lipofectamine twice with 48 h intervals increased the efficiency of transfection about 60.21 ± 1.05 % and 65.06 ± 0.09 % for MSCs and DCs, respectively. Moreover, DCs and MSCs transfected with MPER-V3 DNA in heterologous DC or MSC prime/ peptide boost immunizations induced high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses as well as an increased level of Granzyme B. Indeed, the modified MSCs and DCs with MPER-V3 DNA could significantly enhance the MPER/V3-specific T-cell responses compared to MPER/V3 peptide immunization. CONCLUSIONS: These findings showed that the modified MSC-based immunization could elicit effective immune responses against HIV antigen similar to the modified DC-based immunization.

Efficacy of mechanical vibration in regulating mesenchymal stem cells gene expression.

This study aimed at investigating the expression of osteoblast and chondrocyte-related genes in mesenchymal stem cells (MSCs), derived from rabbit adipose tissue, under mechanical vibration. The cells were placed securely on a vibrator's platform and subjected to 300 Hz of sinusoidal vibration, with a maximum amplitude of 10 µm, for 45 min per day, and for 14 consequent days, in the absence of biochemical reagents. The negative control group was placed in the conventional culture medium with no mechanical loading. The expression of osteoblast and chondrocyte-related genes was investigated using real-time polymerase chain reaction (real-time PCR). In addition, F-actin fiber structure and alignment with the help of actin filament fluorescence staining were evaluated, and the level of metabolic activity of MSCs was determined by the methyl thiazolyl tetrazolium assay. The real-time PCR study showed a significant increase of bone gene expression in differentiated cells, compared with MSCs (P < 0.05). On the other hand, the level of chondrocyte gene expression was not remarkable. Applying mechanical vibration enhanced F-actin fiber structure and made them aligned in a specific direction. It was also found that during the differentiation process, the metabolic activity of the cells increased (P < 0.05). The results of this work are in agreement with the well-accepted fact that the MSCs, in the absence of growth factors, are sensitive to low-amplitude, high-frequency vibration. Outcomes of this work can be applied in cell therapy and tissue engineering, when regulation of stem cells is required.

Graphene oxide incorporated polycaprolactone/chitosan/collagen electrospun scaffold: Enhanced osteogenic properties for bone tissue engineering.

This in vitro study aimed to evaluate the physicochemical and biological activity of the polycaprolactone/chitosan/collagen scaffolds incorporated with 0, 0.5, 3, and 6 wt% of graphene oxide (GO). Using standard tests and MG-63 cells, the characteristics of scaffolds were evaluated, and the behavior of osteoblasts were simulated, respectively. A non-significant decrease in nanofibers diameter was noted in scaffolds with a higher ratio of GO. The hydrophilicity and bioactivity of the scaffold surface, as well as cell attachment and proliferation, increased in correspondence to an increase in GO. The higher ratio of GO also improved the osteogenesis activity. GO increased the degradation rate, but it was negligible and seemed not enough to endanger stability. Modifying the scaffolds with GO did not make a significant change to the antibacterial effect.

An investigation into osteogenic differentiation effects of silk fibroin-nettle (Urtica dioica L.) nanofibers.

The purpose of this study was to investigate physical, mechanical, and osteogenic properties of silk fibroin (SF) nanofibers containing Urtica dioica L. (nettle) extract at different concentrations. In this respect, the successful incorporation of nettle in SF nanofibers was analyzed and then confirmed through Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The mean fiber diameter, water uptake, breaking strain, cellular attachment, and proliferation of the given nanofibers also increased as the nettle content was added, while this trend was opposite in terms of tensile strength and modulus. The in vitro release studies correspondingly demonstrated that the nettle release had been controlled according to Fickian diffusion and it was faster in the samples including more nettle. Furthermore, both ARS staining and real-time RT-PCR results suggested that nettle had enhanced the expression of both early and late markers of osteoblast differentiation in a dose-dependent manner.

A conductive cell-imprinted substrate based on CNT-PDMS composite.

Cell function regulation is influenced by continuous biochemical and biophysical signal exchange within the body. Substrates with nano/micro-scaled topographies that mimic the physiological niche are widely applied for tissue engineering applications. As the cartilage niche is composed of several stimulating factors, a multifunctional substrate providing topographical features while having the capability of electrical stimulation is presented. Herein, we demonstrate a biocompatible and conductive chondrocyte cell-imprinted substrate using polydimethylsiloxane (PDMS) and carbon nanotubes (CNTs) as conductive fillers. Unlike the conventional silicon wafers or structural photoresist masters used for molding, cell surface topographical replication is challenging as biological cells showed extremely sensitive to chemical solvent residues during molding. The composite showed no significant difference compared with PDMS with regard to cytotoxicity, whereas an enhanced cell adhesion was observed on the conductive composite's surface. Integration of nanomaterials into the cell seeding scaffolds can make tissue regeneration process more efficient.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure.

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-ß1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-ß1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.