Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) supports adhesion and migration of mesenchymal stem cells and tenocytes

AIM: To establish the potential of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) as a material for tendon repair. METHODS: The biocompatibility of PHBHHx with both rat tenocytes (rT) and human mesenchymal stem cells (hMSC) was explored by monitoring adhesive characteristics on films of varying weight/volume ratios coupled to a culture atmosphere of either 21% O2 (air) or 2% O2 (physiological normoxia). The diameter and stiffness of PHBHHx films was established using optical coherence tomography and mechanical testing, respectively. RESULTS: Film thickness correlated directly with weight/volume PHBHHx (r2 = 0.9473) ranging from 0.1 mm (0.8% weight/volume) to 0.19 mm (2.4% weight/volume). Film stiffness on the other hand displayed a biphasic response which increased rapidly at values > 1.6% weight/volume. Optimal cell attachment of rT required films of ≥ 1.6% and ≥ 2.0% weight/volume PHBHHx in 2% O2 and 21% O2 respectively. A qualitative adhesion increase was noted for hMSC in films ≥ 1.2% weight/volume, becoming significant at 2% weight/volume in 2% O2. An increase in cell adhesion was also noted with ≥ 2% weight/volume PHBHHx in 21% O2. Cell migration into films was not observed. CONCLUSION: This evaluation demonstrates that PHBHHx is a suitable polymer for future cell/polymer replacement strategies in tendon repair.

Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor （bFGF） gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid plRES2-EGFP to construct eukaryotic expression plasmid plRES2-EGFP-bFGF. The plRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the plRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that plRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

PRELIMINARY STUDY ON IN VITRO TENDON ENGINEE-RING USING TENOCYTES AND POLYGLYCOLIC ACIDS

Objective To investigate the feasibility of tendon engineering in vitro using tenocyws and polyglycolic acids （ PGA ）. Methods Tenocytes were isolated by tissue explant method and expanded in vitro. Cells of the second passage were collected and seeded onto PGA scaffolds made from PGA unwoven fibers at the density of 20 × 10^6 cells/ml. At 1 week postseeding ,the constructs were divided into three groups as follows： cell-scaffold constructs under constant tension generated by a U-shaped spring as the experimental group （ n = 5 ）, cell-scaffold constructs under no tension as control group 1 （ n = 4 ）, cell-free scaffolds under constant tension as control group 2 （n =3）. Samples were harvested at 2, 4 and 6 weeks for histological and immunohistochemical （ IHC ） examinations. Transmission electron microscopy （TEM） and mechanical test were performed to evaluate the constructs of 6 weeks. Results At 2 weeks, the constructs were mainly composed of undegraded PGA fibers. Gross and histological examination revealed no difference between the groups. At 4 weeks, neo-tendon was visible through gross observation in experimental group and control group 1. Histology and immunohistochemistry revealed the formation of collagen fibers. While in control group 2, PGA fibers were mostly degraded. At 6 weeks, the constructs were much thinner in experimental group than those in control group 1 （ 1.44 ± 0.13mm vs 2.55 ± 0. 18mm in diameter ）. TEM showed periodical strata of collagen fibers in the constructs from experimental group and control group 1. However, histology in experimental group revealed longitudinal alignment of collagen fibers, which more resembled natural tendon than neotendon formed in control group 1. Besides, the maximum load to failure（ Newton/mm^2 ） was greater in experimental group than that in control group 1 （1. 107 ±0. 327 vs 0. 294 ± 0. 138, P 〈0.05）. Conclusion It＇ s possible to engineer tendon substitutes in vitro. Cyclic strain generated by a bioreactor may be the optimal mechanical stimulation and is currently under investigation.

In silico analysis of RNA and small RNA sequencing data from human BM-MSCs and differentiated osteocytes, chondrocytes and tenocytes

Background:Adult stem cells have a remarkable capacity of differentiating into various cell types necessary for tissue and organ regeneration.Multiple studies have focused on the differentiation potential of mesenchymal stem cells(MSCs),however little is known about the molecular characteristics of MSCs and their progenies obtained from donors of different ages.In this study,we analyzed publicly available sequencing data obtained from young(~22-year-old,n=8)and older(~65.5-year-old,n=8)donors of MSCs and their differentiated counterparts:osteocytes,chondrocytes and tenocytes.The raw mRNA and small RNA(non-coding RNA)sequencing data was downloaded from NIH BioProjects and systematically analyzed in order to identify uniquely expressed genes in MSC-derived osteocytes,chondrocytes and tenocytes of younger and older people.Results:We identified many commonly up-and downregulated genes are similar in both groups.However,the young group displayed a greater variety of differentially expressed genes in all analyzed MSC-derived cells.This discrepancy in gene expression profiles between younger and older groups may indicate a greater differentiation potential of MSCs isolated from younger donors.miRNA and mRNA integrated analysis showed key miRNAs that regulate mRNAs in both groups from all differentiated lineages.Conclusions:Our analysis provides additional information to previously reported data for identification of MSC markers of plasticity and engraftment.In addition,our data may shed light upon the molecular mechanisms of age-associated musculoskeletal diseases caused by a decreased capacity of MSCs to regenerate the locomotor system in elderly people.

Control of tendon cell fate in the embryonic limb: A molecular perspective

The molecular cascade underlying tendon formation starts when progenitor cells begin to express the Scleraxis(Scx)gene.Scx knockout mice develop some but not all tendons,suggesting that additional factors are necessary for tendon commitment,maintenance,and differentiation.Other transcription factors,such as Mohawk(Mkx)or early growth response(Egr),maintain Scx expression and extracellular matrix formation during fibrillogenesis.The inhibition of wingless and int-related protein signaling is necessary and sufficient to induce the expression of Scx.Once the commitment of tenogenic lineage occurs,transforming growth factor-beta(TGFβ)induces the Scx gene expression,becoming involved in the maintenance of tendon cell fate.From this point of view,we discussed two phases of the tenogenic process during limb development:dependent and independent of mechanical forces.Finally,we highlight the importance of understanding embryonic tendon development to improve therapeutic strategies in regenerative medicines for tendons.

Understanding cellular and molecular mechanisms of pathogenesis of diabetic tendinopathy

There is accumulating evidence of an increased incidence of tendon disorders in people with diabetes mellitus.Diabetic tendinopathy is an important cause of chronic pain,restricted activity,and even tendon rupture in individuals.Tenocytes and tendon stem/progenitor cells(TSPCs)are the dominant cellular components associated with tendon homeostasis,maintenance,remodeling,and repair.Some previous studies have shown alterations in tenocytes and TSPCs in high glucose or diabetic conditions that might cause structural and functional variations in diabetic tendons and even accelerate the development and progression of diabetic tendinopathy.In this review,the biomechanical properties and histopathological changes in diabetic tendons are described.Then,the cellular and molecular alterations in both tenocytes and TSPCs are summarized,and the underlying mechanisms involved are also analyzed.A better understanding of the underlying cellular and molecular pathogenesis of diabetic tendinopathy would provide new insight for the exploration and development of effective therapeutics.

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.