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Physical and Natural Crosslinking Approaches on Three-Dimensional Gelatin Microspheres for Cartilage Regeneration

Sulaiman S, Rani RA, Mohamad Yahaya NH, Tabata Y, Hiraoka Y, Seet WT, et al.

Tissue Eng Part C Methods, 2022 10;28(10):557-569.
PMID: 35615885 DOI: 10.1089/ten.TEC.2022.0073

The use of gelatin microspheres (GMs) as a cell carrier has been extensively researched. One of its limitations is that it dissolves rapidly in aqueous settings, precluding its use for long-term cell propagation. This circumstance necessitates the use of crosslinking agents to circumvent the constraint. Thus, this study examines two different methods of crosslinking and their effect on the microsphere's physicochemical and cartilage tissue regeneration capacity. Crosslinking was accomplished by physical (dehydrothermal [DHT]) and natural (genipin) crosslinking of the three-dimensional (3D) GM. We begin by comparing the microstructures of the scaffolds and their long-term resistance to degradation under physiological conditions (in an isotonic solution, at 37°C, pH = 7.4). Infrared spectroscopy indicated that the gelatin structure was preserved after the crosslinking treatments. The crosslinked GM demonstrated good cell adhesion, viability, proliferation, and widespread 3D scaffold colonization when seeded with human bone marrow mesenchymal stem cells. In addition, the crosslinked microspheres enhanced chondrogenesis, as demonstrated by the data. It was discovered that crosslinked GM increased the expression of cartilage-related genes and the biosynthesis of a glycosaminoglycan-positive matrix as compared with non-crosslinked GM. In comparison, DHT-crosslinked results were significantly enhanced. To summarize, DHT treatment was found to be a superior approach for crosslinking the GM to promote better cartilage tissue regeneration.

Matched MeSH terms: Chondrogenesis
Fulltext In vitro chondrogenesis transformation study of mouse dental pulp stem cells

Zainal Ariffin SH, Kermani S, Megat Abdul Wahab R, Senafi S, Zainal Ariffin Z, Abdul Razak M

ScientificWorldJournal, 2012;2012:827149.
PMID: 22919354 DOI: 10.1100/2012/827149

A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

Matched MeSH terms: Chondrogenesis*
Effects of different media on the in vivo chondrogenesis of sheep bone marrow stem cells: histological assessment

Alfaqeh H, Chua KH, Aminuddin BS, Ruszymah BH

Med J Malaysia, 2008 Jul;63 Suppl A:119-20.
PMID: 19025014

This study aimed to compare the effects of three different media on the in vivo chondrogenesis of sheep bone marrow stem cells (BMSC). Sheep BMSC were cultured in F12:DMEM + 10% FBS, chondrogenic medium containing 5ng/ml TGF,3 + 50ng/ml IGF-1 and UKM-MECC for three weeks. The cultured cells were then harvested for construct formation with fibrin. Constructed tissues were implanted subcutaneously into nude mice for in vivo development. Cell aggregates were formed in both chondrogenic medium and UKM-MECC demonstrated the early chondrogenesis process. After five weeks of in vivo development, both chondrogenic medium and UKM-MECC promoted cartilage matrix synthesis confirmed by Safranin O staining.

Matched MeSH terms: Chondrogenesis/physiology*
Scaffolds for Cartilage Regeneration: To Use or Not to Use?

Sha'ban M, Ahmad Radzi MA

Adv Exp Med Biol, 2020;1249:97-114.
PMID: 32602093 DOI: 10.1007/978-981-15-3258-0_7

Joint cartilage has been a significant focus on the field of tissue engineering and regenerative medicine (TERM) since its inception in the 1980s. Represented by only one cell type, cartilage has been a simple tissue that is thought to be straightforward to deal with. After three decades, engineering cartilage has proven to be anything but easy. With the demographic shift in the distribution of world population towards ageing, it is expected that there is a growing need for more effective options for joint restoration and repair. Despite the increasing understanding of the factors governing cartilage development, there is still a lot to do to bridge the gap from bench to bedside. Dedicated methods to regenerate reliable articular cartilage that would be equivalent to the original tissue are still lacking. The use of cells, scaffolds and signalling factors has always been central to the TERM. However, without denying the importance of cells and signalling factors, the question posed in this chapter is whether the answer would come from the methods to use or not to use scaffold for cartilage TERM. This paper presents some efforts in TERM area and proposes a solution that will transpire from the ongoing attempts to understand certain aspects of cartilage development, degeneration and regeneration. While an ideal formulation for cartilage regeneration has yet to be resolved, it is felt that scaffold is still needed for cartilage TERM for years to come.

Matched MeSH terms: Chondrogenesis*
Growth medium with low serum and transforming growth factor beta 3 promotes better chondrogenesis of bone marrow-derived stem cells in vitro and in vivo

Alfaqeh HH, Hui CK, Saim AB, Idrus RB

Saudi Med J, 2011 Jun;32(6):640-1.
PMID: 21666950
Matched MeSH terms: Chondrogenesis*
Fulltext Mesenchymal stromal cells for cartilage repair in osteoarthritis

Mamidi MK, Das AK, Zakaria Z, Bhonde R

Osteoarthritis Cartilage, 2016 Aug;24(8):1307-16.
PMID: 26973328 DOI: 10.1016/j.joca.2016.03.003

Treatment for articular cartilage damage is quite challenging as it shows limited repair and regeneration following injury. Non-operative and classical surgical techniques are inefficient in restoring normal anatomy and function of cartilage in osteoarthritis (OA). Thus, investigating new and effective strategies for OA are necessary to establish feasible therapeutic solutions. The emergence of the new discipline of regenerative medicine, having cell-based therapy as its primary focus, may enable us to achieve repair and restore the damaged articular cartilage. This review describes progress and development of employing mesenchymal stromal cell (MSC)-based therapy as a promising alternative for OA treatment. The objective of this review is to first, discuss how in vitro MSC chondrogenic differentiation mimics in vivo embryonic cartilage development, secondly, to describe various chondrogenic differentiation strategies followed by pre-clinical and clinical studies demonstrating their feasibility and efficacy. However, several challenges need to be tackled before this research can be translated to the clinics. In particular, better understanding of the post-transplanted cell behaviour and learning to enhance their potency in the disease microenvironment is essential. Final objective is to underscore the importance of isolation, storage, cell shipment, route of administration, optimum dosage and control batch to batch variations to realise the full potential of MSCs in OA clinical trials.

Matched MeSH terms: Chondrogenesis
Immunomodulatory Effect of Cytokines in the Differentiation of Mesenchymal Stem Cells: A Review

Zahari W, Hashim SN, Yusof MF, Osman ZF, Kannan TP, Mokhtar KI, et al.

Curr Stem Cell Res Ther, 2017;12(3):197-206.
PMID: 27306400 DOI: 10.2174/1574888X11666160614103404

Mesenchymal stem cells (MSCs) are stromal origin cells with multilineage differentiation capacity. The immunoregulatory properties of MSCs can be interfered effectively by cytokines. Cytokines, produced by a broad range of cells, act at the systemic level to influence biological phenomena such as inflammation, wound healing, organogenesis and oncogenesis. Cytokines also play vital roles in the differentiation of MSCs into several cell lineages. This review summarizes on how cytokines can affect MSCs differentiation and their relative signaling pathways, which may serve to understand the possible underlying mechanisms. Also, this review reveals the potential clinical use of MSCs as promising therapeutic agents due to their special characteristics such as multipotent differentiation, immunomodulatory properties, and selfrestoration.

Matched MeSH terms: Chondrogenesis/genetics; Chondrogenesis/immunology*
Fulltext Proteomics of chondrogenesis: a review

Tay, L.X.

JUMMEC, 2015;18(1):1-8.
MyJurnal

Osteoarthritis (OA) affects millions of people worldwide with its irreversible destruction of articular cartilage. Recently, the potential of using chondrogenic differentiated multipotent mesenchymal stromal cells (cMSCs) for OA treatment is being assessed. Preliminary clinical studies have been encouraging. However current studies have also demonstrated that cMSCs are not biochemically and biomechanically identical to native articular chondrocytes (ACs). Thus, there is an urgent need for the implementation of proteomic applications as proteomics involve protein identification, relative quantification of proteins and studies of post-translational modification which reveal novel regulating processes of complex mechanisms such as in chondrogenesis. A comprehensive understanding of chondrogenesis is essential for the establishment of an effective cMSC model to regenerate cartilage. In this article, we will review current proteomic studies on chondrogenesis, focusing on recent findings and the proteomic approaches utilised.

Matched MeSH terms: Chondrogenesis
Fulltext Clinical Potential of Cellular Material Sources in the Generation of iPSC-Based Products for the Regeneration of Articular Cartilage

Eremeev A, Pikina A, Ruchko Y, Bogomazova A

Int J Mol Sci, 2023 Sep 22;24(19).
PMID: 37833856 DOI: 10.3390/ijms241914408

Inflammatory joint diseases, among which osteoarthritis and rheumatoid arthritis are the most common, are characterized by progressive degeneration of the cartilage tissue, resulting in the threat of limited or lost joint functionality in the absence of treatment. Currently, treating these diseases is difficult, and a number of existing treatment and prevention measures are not entirely effective and are complicated by the patients' conditions, the multifactorial nature of the pathology, and an incomplete understanding of the etiology. Cellular technologies based on induced pluripotent stem cells (iPSCs) can provide a vast cellular resource for the production of artificial cartilage tissue for replacement therapy and allow the possibility of a personalized approach. However, the question remains whether a number of etiological abnormalities associated with joint disease are transmitted from the source cell to iPSCs and their chondrocyte derivatives. Some data state that there is no difference between the iPSCs and their derivatives from healthy and sick donors; however, there are other data indicating a dissimilarity. Therefore, this topic requires a thorough study of the differentiation potential of iPSCs and the factors influencing it, the risk factors associated with joint diseases, and a comparative analysis of the characteristics of cells obtained from patients. Together with cultivation optimization methods, these measures can increase the efficiency of obtaining cell technology products and make their wide practical application possible.

Matched MeSH terms: Chondrogenesis
Growth kinetic study on normal and microtic chondrocytes of human auricular cartilage

Ishak MF, Aminuddin BS, Asma A, Lokman BS, Ruszymah BH, Goh BS

Med J Malaysia, 2008 Jul;63 Suppl A:117-8.
PMID: 19025013

Chondrocytes were isolated from normal and microtic human auricular cartilage after ear surgery carried out at Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured and expanded until passage 4. After reached confluence, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and assigned for growth profile analysis. There was no significant difference in cell viability between both normal and microtic samples (p = 0.84). Both samples showed no significant differences for growth profile parameters in terms of growth rate, population doubling time and total number of cell doubling, except in passage 1, where there is significant difference in cell growth rate (p = 0.004). This preliminary data has indicated that chondrocytes from microtic cartilage has the potential to be used in the reconstruction of human pinna in the future.

Matched MeSH terms: Chondrogenesis/physiology*
Three dimensional alginate-fucoidan composite hydrogel augments the chondrogenic differentiation of mesenchymal stromal cells

Karunanithi P, Murali MR, Samuel S, Raghavendran HRB, Abbas AA, Kamarul T

Carbohydr Polym, 2016 08 20;147:294-303.
PMID: 27178935 DOI: 10.1016/j.carbpol.2016.03.102

Presence of sulfated polysaccharides like heparan sulphate has often been implicated in the regulation of chondrogenesis. However, recently there has been a plethora of interest in the use of non-animal extracted analogs of heparan sulphate. Here we remodeled alginate (1.5%) by incorporating fucoidan (0.5%), a natural sulphated polysaccharide extracted from seaweeds to form a composite hydrogel (Al-Fu), capable of enhancing chondrogenesis of human mesenchymal stromal cells (hMSCs). We confirmed the efficiency of fucoidan incorporation by FTIR and EDX analysis. Further, its ability to support hMSC attachment and chondrogenic differentiation was confirmed by SEM, biochemical glycosaminoglycan quantification, real-time quantitative PCR and immunocytochemical analyses of chondrogenic markers Sox-9, Collagen II, Aggrecan and COMP. Effect of Al-Fu hydrogel on hMSC hypertrophy was also confirmed by the downregulation of hypertrophic genes Collagen X and Runx2. This composite scaffold can hence be used as a cartilage biomimetic biomaterial to drive hMSC chondrogenesis and for other cartilage repair based therapies.

Matched MeSH terms: Chondrogenesis*
On-Demand Guided Bone Regeneration with Microbial Protection of Ornamented SPU Scaffold with Bismuth-Doped Single Crystalline Hydroxyapatite: Augmentation and Cartilage Formation

Selvakumar M, Srivastava P, Pawar HS, Francis NK, Das B, Sathishkumar G, et al.

ACS Appl Mater Interfaces, 2016 Feb 17;8(6):4086-100.
PMID: 26799576 DOI: 10.1021/acsami.5b11723

Guided bone regeneration (GBR) scaffolds are futile in many clinical applications due to infection problems. In this work, we fabricated GBR with an anti-infective scaffold by ornamenting 2D single crystalline bismuth-doped nanohydroxyapatite (Bi-nHA) rods onto segmented polyurethane (SPU). Bi-nHA with high aspect ratio was prepared without any templates. Subsequently, it was introduced into an unprecedented synthesized SPU matrix based on dual soft segments (PCL-b-PDMS) of poly(ε-caprolactone) (PCL) and poly(dimethylsiloxane) (PDMS), by an in situ technique followed by electrospinning to fabricate scaffolds. For comparison, undoped pristine nHA rods were also ornamented into it. The enzymatic ring-opening polymerization technique was adapted to synthesize soft segments of PCL-b-PDMS copolymers of SPU. Structure elucidation of the synthesized polymers is done by nuclear magnetic resonance spectroscopy. Sparingly, Bi-nHA ornamented scaffolds exhibit tremendous improvement (155%) in the mechanical properties with excellent antimicrobial activity against various human pathogens. After confirmation of high osteoconductivity, improved biodegradation, and excellent biocompatibility against osteoblast cells (in vitro), the scaffolds were implanted in rabbits by subcutaneous and intraosseous (tibial) sites. Various histological sections reveal the signatures of early cartilage formation, endochondral ossification, and rapid bone healing at 4 weeks of the critical defects filled with ornamented scaffold compared to SPU scaffold. This implies osteogenic potential and ability to provide an adequate biomimetic microenvironment for mineralization for GBR of the scaffolds. Organ toxicity studies further confirm that no tissue architecture abnormalities were observed in hepatic, cardiac, and renal tissue sections. This finding manifests the feasibility of fabricating a mechanically adequate nanofibrous SPU scaffold by a biomimetic strategy and the advantages of Bi-nHA ornamentation in promoting osteoblast phenotype progression with microbial protection (on-demand) for GBR applications.

Matched MeSH terms: Chondrogenesis/drug effects*
Fulltext 3D Culture of MSCs on a Gelatin Microsphere in a Dynamic Culture System Enhances Chondrogenesis

Sulaiman S, Chowdhury SR, Fauzi MB, Rani RA, Yahaya NHM, Tabata Y, et al.

Int J Mol Sci, 2020 Apr 13;21(8).
PMID: 32294921 DOI: 10.3390/ijms21082688

Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation.

Matched MeSH terms: Chondrogenesis*
Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique

Goh BS, Che Omar SN, Ubaidah MA, Saim L, Sulaiman S, Chua KH

Acta Otolaryngol, 2017 Apr;137(4):432-441.
PMID: 27900891 DOI: 10.1080/00016489.2016.1257151

CONCLUSION: In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue.
OBJECTIVE: Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation.
METHODS: Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days.
RESULTS: According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen. The results showed HADSCs test groups (cultured with TGF-β3) displayed chondrocytes-like cells morphology with typical lacunae structure compared to the control group without TGF-β3 after 2 weeks. Additionally, the HADSCs test groups increased in cell viability; an increase in expression of chondrocytes-specific genes (collagen type II, aggrecan core protein, SOX 9 and elastin) compared to the control. This study found that human auricular chondrocytes cells and growth factor had a positive influence in inducing HADSCs chondrogenic effects, in terms of chondrogenic differentiate of feature, increase of cell viability, and up-regulated expression of chondrogenic genes.

Matched MeSH terms: Chondrogenesis*
Fulltext Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields

Parate D, Franco-Obregón A, Fröhlich J, Beyer C, Abbas AA, Kamarul T, et al.

Sci Rep, 2017 08 25;7(1):9421.
PMID: 28842627 DOI: 10.1038/s41598-017-09892-w

Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common to chondrogenesis. Here we document the effects of specified PEMF parameters over mesenchymal stem cells (MSC) chondrogenic differentiation. MSCs undergoing chondrogenesis are preferentially responsive to an electromagnetic efficacy window defined by field amplitude, duration and frequency of exposure. Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (2 mT) exposure to 6 ms bursts of magnetic pulses, at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome and could be attributed to calcium entry after the initial induction. Transient receptor potential (TRP) channels appear to mediate these aspects of PEMF stimulation, serving as a conduit for extracellular calcium. Preventing calcium entry during the repeated PEMF exposure with the co-administration of EGTA or TRP channel antagonists precluded the inhibition of differentiation. This study highlights the intricacies of calcium homeostasis during early chondrogenesis and the constraints that are placed on PEMF-based therapeutic strategies aimed at promoting MSC chondrogenesis. The demonstrated efficacy of our optimized PEMF regimens has clear clinical implications for future regenerative strategies for cartilage.

Matched MeSH terms: Chondrogenesis/radiation effects*
The Combined Effect of Substrate Stiffness and Surface Topography on Chondrogenic Differentiation of Mesenchymal Stem Cells

Wu Y, Yang Z, Law JB, He AY, Abbas AA, Denslin V, et al.

Tissue Eng Part A, 2017 01;23(1-2):43-54.
PMID: 27824280 DOI: 10.1089/ten.TEA.2016.0123

Stem cell differentiation is guided by contact with the physical microenvironment, influence by both topography and mechanical properties of the matrix. In this study, the combined effect of substratum nano-topography and mechanical stiffness in directing mesenchymal stem cell (MSC) chondrogenesis was investigated. Three polyesters of varying stiffness were thermally imprinted to create nano-grating or pillar patterns of the same dimension. The surface of the nano-patterned substrate was coated with chondroitin sulfate (CS) to provide an even surface chemistry, with cell-adhesive and chondro-inductive properties, across all polymeric substrates. The surface characteristic, mechanical modulus, and degradation of the CS-coated patterned polymeric substrates were analyzed. The cell morphology adopted on the nano-topographic surfaces were accounted by F-actin distribution, and correlated to the cell proliferation and chondrogenic differentiation outcomes. Results show that substratum stiffness and topographical cues affected MSC morphology and aggregation, and influenced the phenotypic development at the earlier stage of chondrogenic differentiation. Hyaline-like cartilage with middle/deep zone cartilage characteristics was generated on softer pillar surface, while on stiffer nano-pillar material MSCs showed potential to generate constituents of hyaline/fibro/hypertrophic cartilage. Fibro/superficial zone-like cartilage could be derived from nano-grating of softer stiffness, while stiffer nano-grating resulted in insignificant chondrogenesis. This study demonstrates the possibility of refining the phenotype of cartilage generated from MSCs by manipulating surface topography and material stiffness.

Matched MeSH terms: Chondrogenesis*
Fulltext Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

Dashtdar H, Murali MR, Selvaratnam L, Balaji Raghavendran H, Suhaeb AM, Ahmad TS, et al.

PeerJ, 2016;4:e1650.
PMID: 26966647 DOI: 10.7717/peerj.1650

Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

Matched MeSH terms: Chondrogenesis
Fulltext Gelatin Microsphere for Cartilage Tissue Engineering: Current and Future Strategies

Sulaiman SB, Idrus RBH, Hwei NM

Polymers (Basel), 2020 Oct 19;12(10).
PMID: 33086577 DOI: 10.3390/polym12102404

The gelatin microsphere (GM) provides an attractive option for tissue engineering due to its versatility, as reported by various studies. This review presents the history, characteristics of, and the multiple approaches to, the production of GM, and in particular, the water in oil emulsification technique. Thereafter, the application of GM as a drug delivery system for cartilage diseases is introduced. The review then focusses on the emerging application of GM as a carrier for cells and biologics, and biologics delivery within a cartilage construct. The influence of GM on chondrocytes in terms of promoting chondrocyte proliferation and chondrogenic differentiation is highlighted. Furthermore, GM seeded with cells has been shown to have a high tendency to form aggregates; hence the concept of using GM seeded with cells as the building block for the formation of a complex tissue construct. Despite the advancement in GM research, some issues must still be addressed, particularly the improvement of GM's ability to home to defect sites. As such, the strategy of intraarticular injection of GM seeded with antibody-coated cells is proposed. By addressing this in future studies, a better-targeted delivery system, that would result in more effective intervention, can be achieved.

Matched MeSH terms: Chondrogenesis
Fulltext Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Nam HY, Pingguan-Murphy B, Abbas AA, Merican AM, Kamarul T

Stem Cells Int, 2019;2019:9723025.
PMID: 30918524 DOI: 10.1155/2019/9723025

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

Matched MeSH terms: Chondrogenesis
Chondrogenesis of adipose-derived stem cells with chondrocytes in low serum towards clinical application

Adila A Hamid, Satish Vaarman Jeyabalan, Aleza Omar, Nik Zattil Hanan Mohd Yasin, Wong TL, Liau LL, et al.

Sains Malaysiana, 2018;47:2369-2379.

Currently, fetal bovine serum (FBS) have been widely use in culture media to promote human cell proliferation. However,
the usage of FBS for cell therapy in clinical application was associated with the risk of viral and prion transmission as
well as immune rejection. To provide an option for this risk, this study was conducted to determine the effect of adipose
derived stem cells (ADSCs) co-culture with chondrocyte in promoting cell proliferation and chondrogenesis toward
FBS free condition. ADSCs co-cultured with chondrocyte at the ratio of 1:1, 2:1 and 1:2 were tested. Cell morphology
changes, cell proliferation and gene expression level of stemness (Oct4, FGF-4, Nanog) and chondrogenic (Collagen
Type II, ACP) were assessed. The results showed ADSCs in all co-culture groups changed morphology from fibroblastic
spindle to polygonal shape which resembled chondrocytes. The morphological changes were accompanied with increased
expression of chondrogenic genes; denoted chondrogenesis process. While maintaining expression of stemness genes
indicated continuation of cell proliferation. From the three co-culture groups tested; ADSCs and chondrocytes (1:1 ratio)
have been shown to exert better effects in promoting cell proliferation and chondrogenesis. In conclusion, ADSCs could
replace FBS to grow sufficient number of chondrogenic cells to repair cartilage injury in the near future. Further in vivo
study should be performed to test the effectiveness of this co-culture technique in cartilage injury repair.

Matched MeSH terms: Chondrogenesis

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