Posterolateral Spinal Fusion With Nano-hydroxyapatite/ Collagen/pla Composite And Admscs In A Rabbit Model

Partâ… :The Isolation,Culture,Differentiation and seeding with nHAC/PLA in Vitro of Adipose Derived Mesenchymal Stem cells.Objective: To study the method of isolating and culturing stem cells from rabbit adipose tissue and to determine if ADMSCs harvest from rabbit could differentiate into adipogenic, osteogenic and chondrogenic in vitro.Method: Autologous subcutaneous adipose tissue in the inguinal groove was harvested from mature female New Zealand white rabbits and digested with collagenase. After primary culture in DMEM and expanded to two passages, the cells were incubated in an adipogenic medium,an osteogenic medium or an chondrogenic medium for 2-4 weeks to induce adipogenesis, osteogenesis and chondrogenesis, respectively. Evidences of adipogenic differentiation was confirmed by Oil-Red staining and osteogenic differentiation was detected by a ALP solution, while chondrogenic differentiation was confirmed using the toluidine blue and safranin O staining at acidic pH.P3 of cells was selected to be seeded with scaffolds. These scaffolds were then threaded onto needles that were embedded in the stoppers of spinner flasks. Cell viability was assessed on day 0, 3, 5, 7 by using the Live/Dead kit while cell proliferation was assessed using a methylthiazol tetrazolium assay.Results: ADMSCs can be isolated from rabbit adipose tissue. It exhibited a heterogeneous population of fibroblast like cells morphologically. In adipogenic induction medium,a significant fraction of the cells contained multiple, intracellular lipid-filled droplets that accumulated Oil Red-O at 2 weeks . The osteogenic differentiation of ADMSCs is induced in osteogenic differentiation medium for 2 weeks. It formed an extensive network of dense, multilayered nodules that stained positively for ALP. Calcification of the ECM matrix was assessed using Alizarin Red staining. Consistent with osteogenesis, several red regions (indicative of a calcified ECM) were observed in ADMSCs treated for 4 weeks toward the osteogenic lineage. Chondrogenic differentiation can be induced in vitro using a technique of micromass culture. The ADMSCs micromass were associated with a Toluidine Blue positive ECM. ADMSCs sections expressed the collagen type II which was demonstrated with immunostaining. The rate of cell attachment by spinner flask seeding method is approximately 95%. ADMSCs are able to maintain their viability and proliferation after seeding by spinner flask.Conclusion:1 ADMSCs exhibit favorable qualities including easy isolation, relative abundance, rapid expansion.2 ADMSCs are able to differentiate into adipogenic, osteogenic, chondrogenic cell types respectively.3 The high rate of cell attachment, viability and proliferation can be maintained by spinner flask seeding method.Partâ…¡: Posterolateral Spinal Fusion with Nano-hydroxyapatite/ Collagen/PLA Composite and ADMSCs in a Rabbit Model.Objective: Spinal fusion is routinely performed to treat low back pain caused by degeneration intervertebral disc. Autologous bone graft derived from the iliac crest is the standard procedure used for spinal fusion. However, several shortcomings, including pseudarthrosis, pain, and the need for blood transfusion are known to be associated with the procedure. Our study analyzed the effectiveness of a new mineralized collagen matrix ,nano-hydroxyapatite /collagen/polylactic acid(nHAC/PLA), combined with autologous adipose- derived mesenchymal stem cells(ADMSC) as a graft material for the posterolateral spinal fusion in the rabbit model.Method: Forty rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC/PLA composite group (nHAC/PLA), autologous iliac crest bone mixed with nHAC/PLA composite group (ACB +nHAC/PLA), and nHAC/PLA composite combined with ADMSC (ADMSC+nHAC/PLA). The viability and the proliferation of the ADMSC seeded on the scaffolds were evaluated respectively by Live/Dead kit and MTT assay in vitro. Lumbar posterolateral fusions were assessed by manual palpation, radiographic, histologic, and mechanical strength, and scanning electronic microscopy (SEM) in a 10-week observation. Results: The results showed that the rate of fusion was significantly higher in ACB and ADMSC+nHAC/PLA groups than that in nHAC/PLA and ACB+nHAC/PLA groups. It was not significantly higher in ACB group than that in ADMSC+nHAC/PLA group. From the microstructure analysis of the samples with histologic staining methods, there was more new bone-like tissue formation in ACB and ADMSC+nHAC/PLA groups than that in other two groups at the 10th postoperative week.Result: Forty rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC/PLA composite group (nHAC/PLA), autologous iliac crest bone mixed with nHAC/PLA composite group (ACB +nHAC/PLA), and nHAC/PLA composite combined with ADMSC (ADMSC+nHAC/PLA). Lumbar posterolateral fusions were assessed by manual palpation, radiographic, histologic, and mechanical strength, and scanning electronic microscopy in a 10-week observation.Conclusion: Our study demonstrated the effective impact of nHAC/PLA combined with ADMSC in rabbit posterolateral spinal fusion.