Study Of Cyclic Tensile Stress Stimulation On The Maturation Of TDSCs-P(LLA-CL)-Col Scaffold Constructs For Tendon Tissue Engineering

Background:Tendon injury caused by a variety of trauma and diseases is very common in clinic.There are at least30,000,000patients with tendon injury every year in the world. At present,autogeneic tendon grafting, allogeneic tendon grafting and prosthetic materials are oftenused for repairing the injured tendons. However, these remodeling techniques have theirown disadvantages, such as complications at the donor site, disease transmission,immunological rejection and incomplete repair. Therefore, it is an urgent need in clinic tofind a kind of bioactive transplantation materials with good properties for repair of tendoninjury.In the past two decades, with the development of cell culture technology,transplantation technology and biomaterial science, the clinical medicine has been steppinginto a new phase of “regenerative medicine”, an ideal tendon substitute—tissue-engineeredtendon is likely to solve the repair issue of injured tendons by reconstructing theregenerative tissues similar to native tendons in biomechanics, biochemistry and histology.The elements for construction of tissue-engineered tendons mainly include seed cells,scaffolding materials, mechanical stimulation, growth factors, etc. The construction offunctional tissue-engineered tendons with good bioactivity and mechanical propertiesrequires the seed cells which can be amplified in a large number and can differentiate intomature tendon cells, the biological scaffolding materials which can provide an enoughgrowth space and mechanical properties, and the proper mechanical or chemical stimulation.So far, these issues have not been adequately addressed, which influences the developmentof tendon tissue engineering and restricts the clinical application of tissue-engineeredtendons in the regenerative repair of tendon defects.This study aimed to systematically investigate all issues about the construction offunctional tissue-engineered tendons, and eventually to construct the tissue-engineered tendons with tendon derive stem cells (TDSCs)-poly(L-lactide-co-e-caprolactone)(P(LLA-CL))-Col nanoyarn scaffolds under cyclic tensile stress stimuli and promote theregeneration of injured tendons. The study contents included the following aspects: In theselection, differentiation and regulation of seed cells, TDSCs were isolated from rabbitpatellar tendon tissues and cultured, we planned to regulate and induce the tenogenicdifferentiation of TDSCs by cyclic tensile stress so as to provide sufficient seed cells, and tolay the foundation for tendon regeneration. In the tissue-engineered scaffold,P(LLA-CL)-Col composite scaffold was fabricated by a new electrospinning method, whichhad a big three-dimensional (3D) spatial structure beneficial for the growth of seed cells anda certain physiological load function that could be applied for functional tendon scaffold;the scaffold morphologically and structurally mimicked the ECM of native tendon tissues.Finally, we would construct the tissue-engineered tendons through seeding of TDSCs in theP(LLA-CL)-Col scaffold followed by mechanical stimulation, and then evaluate theirregenerative role for repairing the tendon injury in an animal model.This study contains three parts:(1) separation, culture and multi-directionaldifferentiation potential identification of rabbit TDSCs;(2) fabrication of a novel,3D,macroporous, aligned P(LLA-CL)-Col nanoyarn network, detection of its physical andchemical properties as well as biocompatibility, and evaluation of its feasibility andadvantages as tissue-engineered tendon scaffold;(3) evaluation of the effects oftissue-engineered tendons constructed in the bioreactor using TDSCs complexP(LLA-CL)-Col scaffold under the tensile stress stimulation in vitro, evaluation of theeffects of new tendon tissues formed using TDSCs-P(LLA-CL)-Col construct implantedinto the back of nude mice under the natural tensile stress stimulation in vivo, andevaluation of the effects of tissue-engineered tendons in prompting the repair of tendoninjury. This study will eventually build the foundation for the clinical application oftissue-engineered tendons and provide a new direction for the treatment of tendon injury.Part1: Separation and Identification of TDSCsObjective:Seed cells play an important role in the repair process of tissues. Tendon cells are themajor functional cells for repair of tendon injury and they synthesize and secrete extracellular matrix (ECM)(e.g., collagen) and maintain the metabolism of tendon tissues.However, they are terminal cells and thus have no self-renewal capacity. TDSCs have beenfound in the recent years and they originate from tendon tissues. TDSCs are the precursorcells of tendon cells and have a strong differentiation potential, so they can be used aseffective seed cells for tendon regeneration. This part aimed to separate and identify TDSCs,as well as evaluate their cloning and three-lineage differentiation potentials.Methods:Tendon cells were separated by digestion with type I collagenase and dispase. TDSCswere obtained by low-density inoculation (2cells/cm2) and monoclone culture; their cloningpotential was detected by crystal violet staining, their osteogenic, adipogenic andchondrogenic differentiations were induced separately by adding the chemical inductionfactors, and their three-lineage differentiation potentials were detected by0.1%alizarin red,0.5%oil red O, and toluidine blue staining.Results:Tendon cells were separated from rabbit patellar tendon tissues by digestion with type Icollagenase and dispase, and TDSCs were obtained by monoclone culture. The majority ofTDSCs was cobblestoning ellipse, and the minority of TDSCs was polygonal and fusiform.After osteogenic induction culture, the alizarin red staining showed that TDSCs had visiblecalcium nodules; after adipogenic induction culture, the oil red staining showed that TDSCscontained a large number of lipid droplets in the cytoplasm; after chondrogenic inductionculture, the toluidine blue staining showed that TDSCs had a large number of bluemetachromatic granules inside. These findings proved the fact that TDSCs had amulti-directional differentiation potential.Conclusion:TDSCs from tendon tissues have general characteristics of stem cells and may be asource of ideal seed cells for in vitro construction and in vivo tendon injury repair oftissue-engineered tendons. Part2: Fabrication of A New Electrospun P(LLA-CL)-Col NanoyarnTissue-engineered Tendon Scaffold and Investigation of Its PerformanceObjective:Tissue engineering based on new scaffolding materials provides a potential therapeuticstrategy for the treatment of tendon diseases. The frequently-used tissue-engineered tendonscaffolds often fail to well simulate the3D structure and morphology of native tendonmatrix. Electrospinning is a simple, practical method to fabricate the high-porosity,nanometer to micrometer scaffolds with a similar structure to ECM. The frequently-usedelectrospun nanofiber scaffolds have a pore size which is not beneficial for cell ingrowth,while the electrospun nanoyarm has a3D macroporous microstructure composed by loose,disperse nanofibers and thus has improved permeability. In this part, we used P(LLA-CL)with good elasticity, biodegradation property, and certain mechanical strength and type Icollagen which can improve cell adhesion as raw materials, fabricated a3D, macroporous,aligned nanoyarn network with a morphology and structure similar to native tendon tissuematrix and evaluated its feasibility as tissue-engineered tendon scaffold.Methods:A new dynamic water collection system was used to prepare P(LLA-CL)-Col nanoyarnduring the electrospinning process, and the nanofiber scaffold was fabricated with thetraditional method. The morphology, porosity, composition and mechanical properties of thescaffold, the adhesion, proliferation and permeation of tendon cells on the scaffold, and theexpression of tendon-specific ECM genes were evaluated.Results:The P(LLA-CL)-Col nanoyarn scaffold had a3D aligned microstructure, and it had abigger pore size and higher porosity than the random and aligned P(LLA-CL)-Col nanofiberscaffolds. Tendon cells growing on the nanoyarn scaffold demonstrated a better naturalmorphology, better proliferation and more ECM secretion than those growing on therandom and aligned nanofiber scaffolds. Importantly, the spatial structure ofP(LLA-CL)-Col nanoyarn scaffold was conducible to the cell migration into the scaffold,the expression of tendon-related ECM genes, and the formation of high-quality tendon-liketissues. Furthermore, this nanoyarn scaffold exhibited desirable mechanical properties forapplication in the regeneration of tendon tissues.Conclusion:The Electrospun P(LLA-CL)-Col nanoyarn is a novel,3D, macroporous, alignedscaffold. It is very suitable for the growth of seed cells on the surface and in the inside and has a good application prospect in the construction of tissue-engineered tendons.Part3: Investigation on the Biological Effects of Cyclic Tensile Stress inPromoting the Maturation of TDSCs-P(LLA-CL)-Col ComplexObjective:TDSCs are an attractive source of seed cells in tendon injury repair and construction oftissue-engineered tendons. However, the application of these cells in tendon tissueengineering has not been explored fully yet. Cyclic tensile stress plays an important role inthe development and post-injury remodeling of tendons and ligaments. This part aimed toevaluate the effects of tissue-engineered tendons constructed through seeding of TDSCs in aP(LLA-CL)-Col scaffold under the tensile stress stimulation.Methods:The tissue-engineered tendons were cultured for14d in the bioreactor (developed byour research group) under0.5HZ,4%(amplitude),2h/d cyclic tensile stress (experimentalgroup), while those in the control group were subject to static culture. The effects of cyclictensile stress on the viability, proliferation, and tenogenic differentiation mRNA and proteinexpression of TDSCs were evaluated. Two ends of2cm tissue-engineered tendons (totallength:4.5cm) were sutured onto the back ligament of nude mice (mechanical stimulationgroup); the remaining2cm tissue-engineered tendons were implanted subcutaneouslywithout suturing (non-stimulation control group). At2w,4w and8w after operation, the invivo fluorescence imaging, histological and immunohistochemistry, and collagen assaywere performed. Finally the scaffold, dynamically and statically cultured tissue-engineeredtendons were transplanted into rabbit patellar tendon defect sites; at4w and12w afteroperation, the histological and mechanical test were performed.Results:Mechanical stimulation had no effect on the viability of TDSCs, and could promote theproliferation and tenogenic differentiation of TDSCs in vitro. After TDSCs-P(LLA-CL)-Colcomplex was transplanted into nude mice, native mechanical stimulation similarly promotedthe formation of new tendon tissues in vivo. More importantly, mechanical-stimulatedTDSCs-P(LLA-CL)-Col complex could better promote the repair of rabbit patellar tendondefect, and the collagen content and tendon-related protein expression and the mechanical properties of regenerative tendon tissues were significantly increased and improved.Conclusion:TDSCs have a great application potential in the regeneration of injured tendons as seedcells, and the mechanical stimulation is helpful for the maturation of tissue-engineeredtendons. These may provide new strategies for the clinical treatment of tendon injury in thefuture.