Thermosensitive Hydrogels With Bone Marrow Mesenchymal Stem Cells For Repair Of Cartilage Defects

Background:Repair of cartilage defects is an enormous challenge to doctors andscientists. The joint pain and dysfunction caused by the cartilage defectsbright great suffering to the patients. Cartilage is difficult to repairspontaneously as its avascular structure and poor migration ability ofchondrocytes. Articular cartilage belongs to hyaline cartilage which issmooth and elastic to maximize the absorption of buffering stress. One ofcommon used method in clinical is bone marrow stimulation such asdrilling of subchondral bone or microfractures that aims to boost the stemcells into the lesion site. However, the repaired tissues are mainlyfibrocartilage with lower property than that of hyaline cartilage, andultimately leading to osteoarthritis.Cell therapy proves to be promising for cartilage regeneration.Chondrocytes originate from mesenchymal stem cells (MSCs). In recentyears, MSCs become widely used seed cells because of the ability of selfreplication and multi-directional differentiation. In particular, the bonemarrow mesenchymal stem cells (BMMSCs) with convenient extractionin minimal invasive procedure and easy in vitro proliferation are closestto the clinical application. BMMSCs maintain the stable ability todifferentiate into chondrocytes which are available cartilage precursorcells all one’s life. In addition, the high density of BMMSCs in the threedimensional (3D) culture significantly improved chondrogenicdifferentiation than that of adipose-derived mesenchymal stem cells (ADMSCs).As BMMSCs cultured in the three dimensional (3D) scaffolds couldpromote chondrogenesis, a variety of biomaterials have been developedfor cartilage tissue engineering., Type II collagen and proteoglycans form3D structure and are filled with mostly liquid in cartilage, which issimilar to hydrogels with3D networks that could also absorb a lot ofwater. Therefore, hydrogels are at the forefront of cartilage regenerationarea in the last few decades and would continue to lead. Specially,thermosensitive hydrogels have drawn much attention because of theunique injectable property via sol-gel transition at body temperaturewithout any other trigger points. Synthetic thermosensitive hydrogelscould control their properties which is vital important to thebiomechanical loading. Poly(lactide-co-glycolide)(PLGA) is a functionalhydrophobic polymer with good biodegradability, biocompatibility andhas been approved by Food and Drug Administration (FDA). Mostnotable, as the degradation product of PLGA, glutamic acid is the highestcontent of amino acids in articular cartilage. Poly(ethylene glycol)(PEG)is a hydrophilic polymer with high water solubility and innocuity. Basedon these polymers, PLGA PEG PLGA triblock copolymer is apromising thermosensitive scaffold as extracellular matrix with goodbiodegradability and biocompatibility.Material and method：In this study, we first synthesized four kinds of PLGA PEG PLGAtriblock copolymers with various block lengths through Ring-OpeningPolymerization (ROP) of LA and GA using PEG as macroinitiator, andcharacterized their chemical structures and compositions by nuclearmagnetic resonance spectroscopy (1H NMR), fourier transform infrared(FT IR), spectra and gel permeation chromatography (GPC), critical micelle concentration (CMC), transmission electron microscope (TEM)and dynamic laser scattering (DLS). The gelling properties of thehydrogels were tested by the vial of inversion and rheologicalexperimental study. The excellent degradabilities of hydrogels wereconfirmed by in vitro and in vivo degradation experiments. Methylthiazolyl tetrazolium (MTT) and cellular adhesion and proliferationassays demonstrated the low cytotoxicities, good adhesion andproliferation abilities of cells in the materials. We used Chinese whiterabbits (male,2.5Kg) to build full thickness cartilage defects (5mmdiameter, depth4mm), the BMMSCs/PLGA PEG PLGA composites (1×106) transplanted into cartilage defects. The control groups filled with0.1mL gel alone. The blank groups underwent no treatment. Grossobservation of cartilage regeneration at postoperative12weeks, thecartilage was resected and each sample was divided into two parts: onepart was used for histological and immunohistochemical evaluation, theother part was used to perform the biomechanical and biochemical tests.Results：PLGA PEG PLGA triblock copolymers were synthesized throughrandom ROP of LA and GA using PEG as macroinitiator and Sn(Oct)2ascatalyst, and verified their chemical structures. Three kinds of thehydrogels (20wt%) in PBS were liquid at low temperature (4°C) andtranslated into gel at body temperature (37°C). The average molecularweight of PLGA was1600g mol-1,880g mol-1,1400g mol-1and1750gmol-1, respectively. LA/GA (mol/mol) was75/25. The CMC data of fourpolymers were6.6mg·L-1,4.2mg·L-1,2.0mg·L-1and1.2mg·L-1,respectively. The hydrodynamic radius of the micelles were32.1±6.9nm,76.3±5.45nm,95.6±7.89nm,148.7±10.13nm. Rheological testsconfirmed the strong mechanical properties of the hydrogels, the G’ of copolymers in PBS were up to about3.0kPa as the increase oftemperature, which highlighted the potential of this material as aninjectable hydrogel for cartilage tissue engineering. The favorabledegradability of the hydrogels was confirmed by in vitro and in vivodegradation experiment, and the degradation rate in vitro degradation arefaster than in vivo degradation. MTT assay revealed cell survival ratemore than90%after72h culture, which showed good biocompatibility.Cellular adhesion and proliferation showed that the relative cellularadhesion rate more than50%on the top of hydrogel, the relative cellularproliferation rate up to several times in the interior hydrogel. Theyconfirmed the prepared hydrogel has excellent cellular adhesion andproliferation abilities. The good cellular and tissular compatibilities ofthermogel were demonstrated. Gross observations found that theregenerated cartilage integrated well with its surrounding normal cartilageand subchondral bone at12weeks post-operation. The similar accumulationof type II collagen and glycosaminoglycans in the repaired cartilage as in thehyaline cartilage were confirmed by immunohistochemical and toluidine bluestaining and biochemical evaluation. The biomechanical properties wereclose to that of normal cartilage by the cyto-nanoindentation tests.Conclusion:PLGA PEG PLGA thermosensitive hydrogels as injectablebiomaterials can be used in tissue engineering and other fields, whichmay be clinically used to promote repair of cartilage defects as BMMSCscarrier.