Objective To investigate the effect of sensory and motor nerve homogenates at different concentrations on the proliferation and osteogenesis differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.Methods The saphenous nerve and the muscle branch of the sciatic nerve in rats were extracted surgically as sensory and motor nerve tissues,respectively.The primary nerve homogenates (10 mg/mL) were prepared as per 10 mg tissue with 1 mL osteoblast inducing conditional media,and 10 times diluted after filtration purification to prepare sensory and motor nerve homogenates at concentration gradients of 1.0,0.1,0.01,0.001 and 0.0001 mg/mL.Cultivation GFP ± rat pups BMSCs in vitro were trained to P3 generation.The experiment was carried out in 3 groups.The sensory and motor nerve homogenates of 500 μL at the above 6 concentration gradients were added during cultivation respectively in the sensory nerve group (n =18) and the motor nerve group(n =18) while 500 μL of osteoblast inducing conditional media was added in the control group(n =3).Cell proliferation quantity detection and alkaline phosphatase (ALP) staining were used to assess the proliferation and differentiation of BMSCs after 14 days.Results According to the results of CCK-8,the cellular absorbance values at concentrations of 1.0 and 0.1 mg/mL homogenate in the sensory nerve group (1.957 ±0.065 and 1.751±0.073) were significantly greater than in the control group (1.145±0.087) while the cellular absorbance value at concentration of 10.0 mg/mL homogenate in the motor nerve group (0.304 ± 0.619) was significandy smaller than in the control group (1.145 ± 0.087) (P ＜ 0.05).According to the ALP staining,the amounts of cellular calcium nodules in the sensory and motor nerve groups (2.667 ± 0.816 and 3.000 ± 0.632,respectively) were significantly smaller than in the control group (11.833 ± 1.471) (P ＜ 0.05).Conclusion Sensory nerve homogenate is different from motor nerve homogenate in that it may promote proliferation of BMSCs and inhibit osteogenesis differentiation of BMSCs in a certain rage of concentrations.

Objective To investigate the effect of prevascularized tissue-engineered bone graft on regeneration of femoral bone defects in rats.Methods Models of femoral bone defect were created at the bilateral hind limbs of 20 healthy female 10 week-old rats which were divided into 2 even groups randomly (n =10).In group A,conventional tissue-engineered bone grafts were transplanted into the femoral bone defects;in group B,tissue-engineered bone grafts and vascular bundles were implanted into the femoral defects.At 1,4 and 8 weeks after operation,3 rats were sacrificed each time in each group to harvest samples.The remaining one in each group served as a spare animal.Regeneration of bone defects and degradation of scaffolds were assessed by radiologic modality and hematein eosin staining.Results At week 1,the new bone ratio (BV/TV) was 5.47％ ± 1.90％ in group A and 8.49％ ± 1.26％ in group B,showing no significant difference (P ＞ 0.05);at weeks 4 & 8,the BV/TV were 17.54％ ±2.04％ and 39.73％ ± 4.01％ in group A,significantly lower than those in group B (25.32％ ± 2.15％ and 53.22％ ± 2.94％) (P ＜ 0.05).At weeks 1 & 4,the scaffold degradation ratios (RSV/SV) were 97.33％ ± 2.52％ and 80.60％ ±4.00％,showing no significant differences from those in group B (95.67％ ±3.51％ and 75.22％ ±6.20％) (P ＞ 0.05).At week 8,the scaffold degradation ratio in group A (65.46％ ±4.51％) was significantly higher than that in group B (50.19％ ±4.91％) (P ＜ 0.05).At week 8,hematein eosin staining showed better integration of scaffolds with the femur,faster degradation of the interior scaffolds and greater osteogenetic activity in group B.Conclusion Prevascularization of tissue-engineered bone graft may increase new bone volume and scaffold degradation rate,promoting repair of femoral bone defects in rats.

Objective To systematically evaluate the biomechanical recovery of drilled holes in the femur in SD rats.Methods Eighteen female SD rats were randomized into 3 even groups (n =6).Models of 2-mm drilled holes in bilateral femurs were established in groups A and B with 2 holes on each side while no drilling was performed in group C.Samples were harvested in group A at postoperative 4 weeks,in group B at postoperative 8 weeks while at both 4 and 8 weeks in group C.The samples were evaluated in terms of linear elasticity (compression test),viscoelasticity (relaxation and creep tests) and durability (fatigue failure test).Micro-CT scan was performed to measure the bone volume fraction (BV/TV) and bone mineral density (BMD) of new bone.Sirus red staining was performed to measure regeneration of type Ⅰ collagen of new bone.Results The elasticity modulus,maximum load,compression strength and conditional yield limit in groups A were significantly lower than those in group B which were also significantly lower than those in group C (P ＜ 0.05).At 7,200 s,the relaxation (14.56 ±0.69 MPa) and creep variation (11.37％ ± 0.70％) in group A were significantly higher than those in group B (11.06 0.63 MPa and 8.98％ ± 0.40％) which were also significantly higher than those in group C (6.99 ±0.56 MPa and 5.10％ ±0.23％) (P ＜ 0.05).At the constant amplitude loads from 20 N to 200 N,from 20 N to 300 N and from 20 N to 400 N,the recycling numbers in group A (6,044.3 ±879.7,4,093.3 ±628.5 and 1,919.3 ±847.5) were significantly lower than those in group B (10,192.3 ± 1,109.1,6,750.6 ± 818.0 and 3,376.6 ± 671.3) which were also significantly lower than those in group C (28,068.3 ±2,702.6,11,788.3 ± 1,141.6 and 5,296.3 ± 735.0) (P ＜ 0.05).By micro-CT scan,the BVT and BMD in group A were significantly lower than those in group B which were also significantly lower than those in group C (P ＜ 0.05).The sirus red staining showed the type Ⅰ collagen in the bone defect area was completely regenerated in group B.Conclusion Systematic biomechanical measurements may actually detect the characteristics of biomechanical recovery of bone holes in SD rats,enriching the basic research on the bone damage repairing progress.

Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.