Preparation Of Injectable Porous Microcarriers Based On Poly(L-glutamic Acids) And Poly(γ-benzyl-L-glutamate) For Application In Tissue Engineering

Bone and cartilage tissue damage caused by defects, injuries or other types ofdamage remain the main obstacles for repair of challenging defects. Recently, tissueengineering (TE) open new perspectives for treatment of tissue damage. A majorproblem in TE is the availability of a suffcient number of cells with the appropriatephenotype for delivery to damaged or diseased cartilage and bone. The microcarrierssystem offers an attractive method for cell amplifcation and enhancement ofphenotype expression. Moreover, the microcarriers allow for easy manipulation orminimally invasive procedures by surgeons, thereby reducing complications andimproving patient comfort and satisfaction. However, injectable microcarrier for invivo tissue regeneration research is still in its infancy. In the present work, on thebasis of current status at home and abroad, porous microcarriers based on syntheticpolypeptide of Poly-L-glutamate acids (PLGA) and Poly-benzyl-L-glutamate (PBLG)were prepared by freezing phase separation technique, double emulsion method andbiomimetic mineralization, respectively. Furthermore, we also tried theirapplications in tissue engineering.Chitosan microspheres were firstly developed by freezing phase separationtechnique. Then, a novel kind of porous PLGA/chitosan polyelectrolyte complex(PEC) microsphere was developed through the electrostatic interaction betweenPLGA and chitosan. The effects of freezing temperature and CS concentration onpore size and porosity of CS microsphere were investigated, respectively, and foundthat the morphologies of products transite from the non-spherical to spherical withthe CS concentration increased from1%to3%. When the CS concentration waselevated to be at2%,2.5%,3%(w/v), porous microspheres could be generated, andincreasing the amount of chitosan in the preparation of microspheres resulted insmaller open pores and porosities. When chitosan concentration was fixed at2% (w/v), and freezing temperature at20°C, chitosan microsphere with optical poresize of47.5±5.4μm was developed. The amount and distribution of PLGAassembled on chitosan microsphere were investigated. The results showd that a largeamount of PLGA (110.3μg/mg) was homogeneously absorbed within PECmicrospheres and not significantly changed the pore structure. Fourier transforminfrared spectroscopy, thermal gravimetric analysis and zeta-potential analyzerrevealed that the PEC microspheres were successfully prepared through electrostaticinteraction.Attachment as well as proliferation of chondrocyte loaded on CS and PECmicrocarriers were examined by confocal laser scanning microscopy and SEM at1,3,5,7days after seeding, respectively. Compared with the microspheres fabricatedby chitosan, the porous PEC microspheres were shown to effciently promotechondrocyte attachment and proliferation and were found to produce significantmore chondrogenic matrix and to retain phenotype expression. Cell numbers withinthe microspheres at1,3,5,7and14days post-seeding were quantified by MTTassay and the expression of aggrecan and collagen type Ⅱ genes was detected byRT-PCR analysis. It can be found that the presence of PLGA helps to retain thechondrocyte phenotype. To further answer whether seeded cells would infiltrate andsurvive in the inner region of microspheres, harboring cells were subjected toconfocal microscope observation after7days. The results showed that the inoculatedcells could sufficiently infiltrate into the most inner region of PEC microspheres.Microspheres fabricated by chitosan/PLGA or chitosan alone were mixed withchondrocytes and injected subcutaneously in nude mice, respectively. After8weeks,harvested specimens from either PEC groups or chitosan alone group showedcartilage-like tissue. By immunohistochemical and toluidine blue staining,respectively, it was found that typical lacunae inhabited by chondrocytes, withabundant deposition of collagen type Ⅱ and proteoglycans in the neo-generatedcartilage. Furthermore, the average wet weight (55.3±7.1mg) of tissue formed from the PEC microspheres/chondrocytes was39.3%higher than that formed from thechitosan microspheres/chondrocytes. Biochemical quantification showed that tissuegenerated from PEC microspheres/chondrocytes had significantly higher GAG/wetweight (47.6±6.1μg/mg), GAG/DNA ratios(62.3±7.1μg/μg), collagen Ⅱ/wet-weight(99.5±16.1μg/mg)and the collagen Ⅱ/DNA ratios (130.2±17.1μg/μg), which weresignificantly higher than those of the chitosan alone group of40.4%,60.9%,52.1%and74.5%, respectively.Injectable porous micromicrocarriers were prepared based on PBLG. Theeffects of diffirent kinds of porogens on the pore size and porosity of PBLGmicrosphere were investigated, and found that when used gelatin as anthermosensitive porogen, porous PBLG microspheres could be generated. Theeffects of porogen amounts and stirred speeds on the the size and structure of PBLGmicrosphere were also investigated. The results showed that when the gelatin contentwas fixed at6.5%, and stirring speeds400rpm, we are easily to produce highlyporous large PBLG microspheres with an average pore size of41±3.8μm and aporosity of79±4.3%. The in vitro degradability of porous microcarriers wasinvestigated with certain incubation time in phosphate buffered saline (PBS) at37°C.After18weeks of incubation, the porous microspheres disintegrated into smallpieces, and as the molecular weights of PBLG microcarriers increased, thedegradation rate reduced. when the PBLG microspheres with a molecular weight of1.2×10~5lost63.7%of the weight and with a molecular weight of3.0×10~5lost40.5%of their weights, respectively. Porous PBLG microcarriers with a molecular weightof3.0×10~5were injecteded subcutaneously in C57rats with the purpose of studyingrelated to the degradation in vivo. The results showd by HE, masson and SEMimages indicated that PBLG microspheres possess good biocompatibility andcapacity to be biodegradable to naturally-occurring biological products. Overall, thescaffolds were well degraded by the host rats and no abnormal conditions wereobserved during the8weeks of implantation. The signifcant difference between in vitro and in vivo degradation in the remainning weights of the PBLG microcarriersat the end of8weeks refects that the degradation rate of PBLG microcarriers can beaccelerated by particular enzyme of living systems.In order to screen the ultimate cytotoxicity of the prepared porous PBLGmicrocarriers, MTT tests were carried out by means of assessing the cell viability ofCCK8cells cultured with3and7days. Chondrocytes culture on tissue culturepolystyrene (TCPS) without microspheres served as the control. The results showedthat the viability levels are similar, reaching the maximum viability of approximately100%, indicating all of these microspheres were found not to be toxic to cells.Attachment as well as proliferation of chondrocyte loaded on CS and PECmicrocarriers were examined by confocal laser scanning microscopy and SEM at1,3,5,7days after seeding, respectively, and found that with longer culture time, themicrospheres could attach more cells and produce more chondrogenic matrix. Cellnumbers within the microspheres at1,3,5,7and14days post-seeding werequantified by MTT assay and the expression of aggrecan and collagen type Ⅱ geneswas detected by RT-PCR analysis. the porous PEC microspheres were shown toeffciently promote chondrocyte attachment and proliferation and were found toproduce significant more chondrogenic matrix and to retain phenotype expression.Chondrocytes on PBLG microcarriers were examined after cultivation of1,3,5,7days by confocal microscope observation. The results implies that initially attachedcells on the surface gradually migrated towards inner region and cells couldsufficiently infiltrate into the most inner region of PBLG microspheres.Chondrocyte-seeded PBLG microcarriers were mixed with chondrocytes andinjected subcutaneously in nude mice. The inject specimens of chondrocyte-seededporous PBLG composites resulted in new cartilage-like tissue formation in vivo ininject sites at4,8and12weeks after injection subcutaneously. Byimmunohistochemical and toluidine blue staining, respectively, it was found thattypical lacunae inhabited by chondrocytes, with abundant deposition of collagen type Ⅱ and proteoglycans in the neo-generated cartilage. The average tissue wetweight (108±10.1mg) formed from the12weeks group was78.6%higher than thatformed from the8weeks group (60±5.1mg), and263.3%higher than that formedfrom the4weeks group (30±3.5mg). Furthermore, the average wet weight of8weeks group was114%higher than that formed from the control group. Biochemicalquantification showed that tissue generated from PBLG microspheres/chondrocyteshad significantly higher GAG/dried weight of0.296±0.02,0.391±0.012and0.443±0.041g/g, and higher collagen Ⅱ/dried-weight of0.385±0.01,0.414±0.01and0.433±0.03g/g at4,8and12weeks after injection subcutaneously, respectively.The bonelike mineral, carbonate apatite, was successfully used to functionalizeporous PBLG microspheres by a biomimetic mineralization method. Moreover, thetime of the bonelike apatite formation on PBLG was proposed. The results showedby SEM and TGA indicated that the amount of mineral phase increased graduallywith increasing incubation time. EDS spectra of the apatite coating on PBLGmicrospheres showed that the Ca/P ratio of apatite mineral was19.02formicrospheres incubated for5days and10.78for those incubated for8days. Whenincreased the incubation time, the Ca/P ratio enhanced. When the incubation wasfixed at11,14and17d, the Ca/P ratio of apatite mineral was2.09,2.03and1.669,respectively. These values are similar to Ca/P ratio of biological apatite of5:3. Thechemical structure of the mineral coating was provided by FT-IR and XRD spectra.It was found that mineralized PBLG microspheres displayed characteristic peaksassociated with HA when the microspheres incubated for11days, and thecharacteristic peaks were enhanced at14and17days. Based on TEM analyses, thecrystal structure of apatite particles on PBLG microspheres incubated for11dayswas detected to be nano-needle, which were similar to the apatite in natural bone.The above results indicated that the initial inorganic particles are not HA. Whenprolonged the incubation time, phosphate ions wereion of gradually replaced andintegrated to produce HA. When the microspheres incubated for11days, mineralized PBLG microspheres displayed characteristic crystal structure related toHA, which were perfected at17days. Adipose derived stem cells were cultured onHA/PBLG microcarriers incubated for11days and were induced to undergoosteogenic differentiation in the presence of HA/PBLG microcarriers for bone tissueengineering. The results showed that HA/PBLG microcarriers is favorable for thegrowth of adipose derived stem cells before and after osteogenic. The observedresults proved that HA/PBLG microcarriers may be promising scaffolding materialsfor bone tissue engineering and regeneration.