The Study On The Degradation And Biocompatibility Of Aligned Nanoscaffolds For Tissue Engineering

An important reason for intervertebral disc herniations or recurrence after operation is that damaged annulus can not naturally repair, and that loses tpreload status and self-stabilizing effect of the intervertebral disc. Both doctors and patients are plagued how to select the operation due to the recurrence of intervertebral disc, although complete discectomy and interbody fusion can solve the problem of relapse, that is not an ideal surgery way,due to its not meeting the physical requirement,and it is forced to do it to some extent. But on the other hand, although a simple excision of herniated nucleus pulposus is more physiological, both doctors and patients are not willing to accept the recurrence rate. Therefore that how to make annulus repair and reduce or eliminate the recurrence of herniated disc is a series of topics in our study group. Development of tissue engineering techniques in bone repair, cartilage repair has made revolutionary progress,but if that can solve the problem of annulus physiological healing?The annulus tissue engineering is more difficult and complex due to annulus’ anisotropy structure, no blood supply, particularity to withstand stress.Aligned nanofiber scaffolds for tissue engineering was prepared by another team in our study group, The aligned scaffolds for silk fibroin/(L-lacticacid-polycaprolactone) (25:75),[SF/P(LLA-CL)] were prepared by electrospinning, and that has been found to have good stess affordability,and the cells could grow into the inside of the scaffolds showing good prospects for engineering.In our study,the degradation behavior and biocompatibility of scaffold were observed and validated using prepared scaffolds.The degradation property and degradation production have an important effection on annulus repair and biomechanical properties meeting physiological requirements.The degradation products and the change of partial microenvironment not only affect the extracellular matrix thereby affect cell proliferaration,diefferentiation and tissue engineering,but also affect biocompatibility. To this end, the study will focus on two aspects.Experiment IExperimental Study on degradation of aligned nanofiber scaffolds for tissue engineering in vitroObjective To study the behavior for aligned nanofiber scaffolds of SF/P(LLA-CL), its change of mechanical properties and the effect of products of degradation on degradation solution.Method Three kinds of aligned nanofiber scaffold [SF, P(LLA-CL)、SF/P(LLA-CL)] were cut into 30mm x 30mm,5 samples from each kind of aligned nanofiber scaffolds, a total of 90 sampls were marked with direction by scanning electron microscope,weighted (M1) then put into a bottle with 20ml of PBS (0.1mol/L, PH 7.4 ± 0.1), and the medium was changed once a month. Three kinds of aligned nanofiber scaffolds were taken out, dried and weighted(MF), at a month, two-month, three-months, four-month, five-month and six- month point repectively. Then the mass loss were calculated according to the formula of ((MI-MF)/MI×100%) and PH value of degradation solution, molecular weight and polydispersity index, mechanical properties were measured at different timeResults With the degradation of aligned nanofiber scffolds,all their PH value, qulity and mechanical Properties declined.1. The mass loss rate of the aligned nanofiber scaffold of SF/P (LLA-CL) at 1-6-moth point was 1.600±0.110%,6.433±0.082%,10.183±0.147%,11.650±0.187%, 23.267±0.294,29.017±0.293% respectively; The mass loss rate of the aligned nanofiber scaffold of P (LLA-CL) was 3.517±0.075%,10.217±0.117%,21.45±0.635%, 27.217±0.232%,44.083±4.127%,50.883±1.963% respectively; The mass loss rate of the aligned nanofiber scaffold of SF was 1.033±0.103%,1.967±0.113%,2.233±0.103%, 2.467±0.052%,2.900±0.126%,4.883±0.264% respectively.2. The PH value of degradation solution of aligned nanofiber scaffold of SF/P (LLA-CL) at 0-6-month observation points was 7.403±0.014,7.403±0.005,7.403±0.005, 7.218±0.010,6.963±0.012,6.753 ±0.010,6.860± 0.009 respectively; The PH value of degradation solution of aligned nanofiber scaffold of P (LLA-CL) was 7.404± 0.005,7.238 ± 0.008,7.010± 0.009,6.445±0.014,6.287± 0.010,5.870± 0.009,6.153±0.008 respectively; The PH value of degradation solution of aligned nanofiber scaffold of SF was 7.403± 0.014,7.415±0.005,7.415±0.005,7.405 ±0.005,7.405±0.008,7.350±0.020.3. The molecular weight of aligned nanofiber scaffold of P (LLA-CL) before the degradation and at 1 month,2-month,3-month,and 6-month point was 33.34±3.45,13.21± 1.25,5.23±0.22,2.24±0.28,1.32±0.35 respectively,and polydispersity of P (LLA-CL) was 33.34±3.45,13.21±1.25,2.24±0.28,1.32±0.35.4. Mechanical properties of the alignment direction:(1) Elongation at break of aligned nanofiber scaffold of SF/P (LLA-CL) before and at 1-4-month observation point was 70.62±14.24%,20.25±4.34%,9.24±3.21,5.23±1.14% and 4.75±1.23% respectively. That of aligned nonofiber scaffold of P (LLA-CL) was 80.32±15.24%, 30.21±5.25%,8.21±2.35%,—, — respectively; Aligned nonofiber scaffold of SF was 3.83±0.40%,2.85±0.32%,1.05±0.14%,1.04±0.13%,0.85±0.14% respectively.(2) The tensile strength of aligned nanofiber scaffold of SF/P (LLA-CL) before and at 1-4-month observation point was 15.60±2.14 Mpa,8.14±1.34 Mpa,5.21±1.23 Mpa, 3.32±0.26 Mpa,1.82±0.42 Mpa respectively; That of aligned nonofiber scaffold of P (LLA-CL) was 20.01±2.87 Mpa,9.21±1.04 Mpa,2.21±0.34 Mpa,—, —respectively; That of aligned nonofiber scaffold of SF was 6.71±0.58 Mpa,4.25±0.23 Mpa,3.15±0.21 Mpa,2.54±0.45 Mpa,1.31±0.46 Mpa. B.The mechanical properties of direction vertical to alignment:(1) Elongation at break of aligned nanofiber scaffold of SF/P (LLA-CL) before and at 1-4-month observation point was 50.62±13.24%,24.15±4.15%,10.25± 1.23%,4.12±0.25%,1.75±0.26% respectively; That of aligned nonofiber scaffold of P (LLA-CL) was 60.32±10.24%,20.15±6.85%,8.42±8.42,—,—respectively; That of aligned nonofiber scaffold of SF was 2.24±0.31%,1.15±0.21%,0.85±0.12%,0.75± 0.13%,0.50±0.12%;(2) The tensile strength of aligned nanofiber scaffold of SF/P (LLA-CL) before and at 1-4-month observation point was 2.06±0.14 Mpa,1.43±0.16 Mpa,1.21±0.21 Mpa,0.85±0.13 Mpa,0.50±0.08Mpa respectively; That of That of aligned nonofiber scaffold of P (LLA-CL) was 2.51±0.24 Mpa,1.32±0.16 Mpa,0.62±0.15 Mpa,—,—respectively; that of That of aligned nonofiber scaffold of SF was 0.87±0.13 Mpa,0.56±0.14 Mpa,0.45±0.15 Mpa,0.32±0.12 Mpa,0.20±0.08 Mpa respectively.Conclusion 1.Repair of annulus of intervertebral disc by aligned anofiber scaffold of SF/P (LLA-CL) properly be completed in 4 months; 2. Compared to aligned nanofibrous scaffolds P (LLA-CL), the nanofibrous scffolds had a lower rate of degradation, better mechanical Properties, and better degradation environment.and that may be relevant to mixed SF.Experiment ⅡExperimental Study on degradation in vivo of aligned nanofiber scaffolds for tissue engineeringObjective To study the behavior for aligned nanofiber scaffolds of SF/P(LLA-CL) and its effect of degradation products to Local microenvironment of body.Methods 42 6-month-old SD rats were randomly divided equally into 7 grous,maked A,B,C,D,E,F,G.The two kinds of aligned nanofiber scaffolds P (LLA-CL) and SF/P (LLA-CL) were randomly implanted subcutaneously in four different parts, each rat was randomly implanted by 2 scaffolds of P (LLA-CL) and 2 scaffolds of SF/P (LLA-CL). Rats from group A,B,C,D,E,F,G were sacrificed by overdose anesthesia at 1 week,1-month,2-month,3-month,4-month,5-month and 6-month point.Then the scaffolds with around soft tissue were taken out for pathology, immunohistochemistry and relative expression of inflammatory gen of necrosis factor -α (TNF-α), transforming growth factor-β (TGF-β),interleukin-1(3 (IL-1β) and interleukin -10 (IL-10) by technology of RT-PCR.. Another 8 6-month-old SD rats were randomly divided equally into 4 groups, marked group H,I,G,K, then the scaffolds mentioned above were randomly implanted subcutaneously in four different parts of each rat containing 2 scaffolds of P(LLA-CL) and 2 scaffolds of SF/P(LLA-CL),the rats in group H,I,QK were were sacrificed by overdose anesthesia at 9-month,12-month,15-month,18-month for observation of final degradation.Results 1.Histology section showed that the aligned nanofiber scaffold of SF/P (LLA-CL) may maintain relatively complete structure in 6 months; 2.The aligned naofiber scaffold swelled and started to layer at 3-month observation point and fragmented at 6-month observation point; 3.Two kinds of scaffolds disappeared at 18-month observation point; 4. The results of Immunohistochemistry and RT-PCR are below..Conclusion Compared to aligned nanofibrous scaffolds P (LLA-CL), the nanofibrous scffolds can maintain its structural integrity for a long time, It was about 18 months for two kinds of scaffold to degradate completely.Experiment IIIExperimental study on basic physicochemical properties and biocompatibility of aligned nanofiberous scaffolds for tissue engineeringObjective To study the basic physical and chemical properties and biocomp-atibility of aligned nanofiberous scaffold SF/P (LLA-CL)Methods 1. In early period the aligned scaffolds of silk fibroin(SF), poly (L-Lactic-co-polycaprolactone)(p(LLA-CL) w:w=50:50)) and silk fibroin/poly(L-Lactic-co-polycaprolactone)(SF/P(LLA-CL) w:w=25:75) were prepared in our study group.The basic physical and chemical properties of the three scaffolds mentioned above were measured including diameter, surface chemical, water contact angle, mechanical properties.2. The rat bone marrow mesenchymal stem cells(BMMS) were extracted, purified, identified then planted in scaffolds mentioned above for adhesion, proliferation essay and also Proliferating Cell Nuclear Antigen (PCNA) of BMSCs for RT-PCR.3. The scaffolds with around soft tissue were removed after the SD rats were sentenced to death at different time points for pathology, immunohistochemistry and The relative gene expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were perfomed.Results 1. C1s, N1s, O1s peak only existed on the X-ray photoelectron spectroscopy of the nanofiber scaffold SF and SF/P (LLA-CL), no Nls peak existed on the X-ray photoelectron spectroscopy of the nanofiber scaffold of P (LLA-CL),and the bond energy corresponding to C1s, N1s, O1s peak were 280ev,390ev,529ev respectively.2. The water contact of aligned nanofiber scaffold SF/P (LLA-CL) was 80 ± 2 degree,its diameter was 345 ± 55nm,the elongation at break of aligned direction was 70.62 ± 14.24%,the tensile strengthen of aligned direction was 15.60 ± 2.14 MPa, the elongation at break of direction vertical to alignment was 50.62 ± 13.24%, the tensile strength of direction vertical to alignment was 2.06 ± 0.14 MPa; The water contact angle of aligned nanofibrous scaffold P (LLA-CL) was 120 ± 3°,the diameter of aligned nanofibrous scaffold P (LLA-CL) was 235 ± 40nm, the elongation at break of alignment direction was 80.32 ± 15.24%, the tensile strength of alignment direction was 20.01 ± 2.87 MPa, the elongation at break of direction vertical to alignment was 60.32 ± 10.24%, the tensile strength of direction vertical to alignment was2.51 ± 0.24 MPa; the water contact of aligned nanofibrous scaffolds SF was 0 degree, the diameter was 220 ± 23nm, the elongation at break of aligned direction was 3.83 ± 0.40%, the tensile strength was 6.71 ± 0.58MPa, the mean elongation at break of direction vertical to alignment was 2.24 ± 0.31%, the tensile strength of direction vertical to alignment was 0.87 ±0.13 MPa.3. The bone marrow mesenchymal stem cells was successfully extracted from the bone marrow of the rat, then purified, identified and induced successfully to form adipogenic and osteogenic cell.and the function of BMSCs on SF/P (LLA-CL) was better than that on P (LLA-CL).4.Compared with the aligned scaffolds SF/P (LLA-CL),the aligned scaffolds p(LLA-CL) more macrophages invaded at 1-and 3-month point,in adddition foreign body giant cells formed at 3-month point.Furthermore the relative expression levels for tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, or interleukin (IL)-1β and IL-10 was higer at different implantation time.Conclusion1. SF in aliged nanofiber scaffold of SF/P (LLA-CL) mainly exist on the its surface;2. Aligned nanofiber scaffold of SF/P (LLA-CL) has mechanical properties similar to annulus;3.the aligned nanofiber scaffold of SF/P (LLA-CL) is better than P (LLA-CL) in main indicators of biocompatibility, which may be related SF distributed over its surface, increasing the cell recognition site, a suitable water the contact angle to promote adhesion proteins and so on.