Research On The Preparation Of Novel Microcavity Hydrogels And Their Application In Cartilage Tissue Engineering

Cartilage is an avascular, no-neural, no immune response and highly specializedconnective tissue. Articular cartilage shows extremely limited capacity to spontaneously healafter degeneration or injury. Cartilage repair remains a great international challenge forresearchers and also for orthopedic surgeons. Nowadays, cartilage tissue engineering (CTE)provides a promising method for cartilage repair and regeneration. However, to obtainlong-term outcome of cartilage repair is one of the current problems. Major obstacles inapplying CTE are the limited number of seed cells and the proper microenvironment.Therefore, it is obvious meaningful to study the functions and effects of novel materials onthe cell growth, ECM secretion, phenotype maintenance and chondrogenesis as well.Hydrogels have good compatibility, mimicking the three-dimensional structure of ECM,proper pore structure, and can provide good mechanical properties for cell adhesion andmigration. But the breakthrough of hydrogel applications in the field of regenerative medicineis still impeded by limited cell growth and affinity sites for subsequent scarce tissueformation.To address this challenge, we proposed a novel microcavitary hydrogel system. Thesystem was an inspiration from an observation of dynamic outgrowth of chondrocytes at thegel edge of conventional cell-laden hydrogel system and this phenomenon of activeproliferation of cells and extracellular matrix (ECM) secretion at the gel edge was hencenamed edge flourish (EF). This EF phenomenon in the traditional hydrogels wasuncontrollable. By mimicking the natural microenvironment structure of cartilage lacuna, wechose sodium alginate hydrogel as cell carrier, combined the gelatin as pore-forming agent,and prepared the novel microcavity structure by producing―edge phenomenon‖in the innerof hydrogel. We aimed to utilize spontaneous formation of micro-cavities to introducemultiple gel edges into the hydrogel bulk, thereby promoting and accommodating theoutgrowth of the proliferating cells from the cell-encapsulating gel phase to themicrocavities-an unconfined phase just like the EF phenomenon. We sought to explore themechanisms of cell proliferation and differentiation within the microcavitary hydrogel for abetter understanding of interaction between cells and materials. Main research work andconclusions are as follows:Firstly, primary chondrocytes were successfully obtained by type II collagen enzymedigestion from the porcine articular cartilage. Primary chondrocytes were cultured in vitro andits biological characteristics were analyzed. The growth curve showed that chondrocytes sub-cultured went through a growth cycle including lag phase, fast growth phase, stationaryphase and growth inhibition. The primary cultured chondrocytes were elliptical shaped andwould gradually change into spindle-shaped or polygon cells after multiple passages. Primarychondrocytes morphology were stained by HE，toluidine blue and safranin O and the stainingresults were close to that of native cartilage. Type II collagen immunohistochemical stainingshowed clear cytoplasm and cell membrane presented with brown. Primary chondrocytesshowed great viability and purity. Therefore, the method for isolating, culturing andidentifying porcine cartilage chondrocytes was successfully established.Secondly, the gelatin microspheres were synthesized based on the double-emulsionmethod. The microspheres showed neat spherical morphology, smooth surface and gooddispensability. The effects of stirring rate on the gelatin microspheres size were investigated.By changing the stirring rate, gelatin microspheres in three different sizes including small size(80-120μm), middle size (150-200μm) and large size (250-300μm) were obtained and threedifferent size microcavity hydrogels were prepared.Based on the established three microcavity hydrogels, we further investigated whetherthe size of microcavity would affect the growth and the function of chondrocytes. Accordingto the data, the encapsulated cells colonies adjacent the gel-cavity interface spontaneouslyoutgrew the hydrogel phase and filled up all cavities. And also cells cultivated inmicrocavitary hydrogel, especially in small size, had preferable abilities of proliferation andhigher expression of cartilaginous markers than in traditional hydrogels. Furthermore, it wasshown by western blot assay that the culture of chondrocytes in microcavitary hydrogel couldimprove the proliferation of cells potentially by inducing the Erk1/2-MAPK pathway. Cells inmicrocavitary alginate hydrogel with pore size within the range of80-120μm were capable ofbetter growth and ECM synthesis, which would provide a preferable microenvironment forchondrocytes.With the superiority of small size microcavity, we further compared this culture systemwith the traditional hydrogels and found that the microcavitary hydrogel exhibitedoutstanding superiorities in helping the dedifferentiated chondrocytes recover the capabilityfor synthesizing cartilaginous ECM. After28days of culture in this microcavitary hydrogel,dedifferentiated chondrocytes regained the ability to produce cartilaginous ECM such as ColII and Aggrecan instead of ColI. The results demonstrated that the progress ofredifferentiation in the microcavitary hydrogel was superior to that in plain alginate hydrogel.We also explored the correlation between chondrocyte redifferentiation in microcavitaryhydrogels and changes in p38and Erk1/2activity. Finally, the effects of RGD peptide immobilization onto microcavity alginate scaffoldsinduced ATDC5chondrogenesis were evaluated. We have therefore covalently modifiedmicrocavity alginate hydrogel with RGD at a percent grafting of1.4%-3.5%. The RGDimmobilized microcavity hydrogel showed good pore connectivity and biocompatibility.Chondrogenic expression levels of the genes were higher in the RGD-immobilized cellconstructs compared to those in microcavitary alginates. After32days of cultivation in vitro,rich-ECM and lots of lacuna was found in the RGD-immobilized cell hydrogels. For the levelof protein, the production of collagen type II and aggrecan was more pronounced. Theobtained results supported the idea that the RGD peptides conjugated to microcavityhydrogels provided a better3D environment for ATDC5adhesion, survival, migration andchondrogenic differentiation. Our results highlighted the importance of RGD participating inthe chondrogenesis.