Introduction: Composites of hydroxyapatite (HA) and biodegradable polymers had been tasted as scaffolds for bone implants with several problems to solve, two of this problems are the cell adhesion and morphology, pore size and accessibility of the HA are important and than the cell interaction with bought materials must be good[1]-[3]. In this work we study a scaffold made by electrospinning from a suspension of HA in polylactic acid (PLA) solutions, to made a material with large porosity and a large surface area. We increased the surface cell interaction by plasma polymerization of pyrrole[4],[5]. Scaffolds were tested with rabbit osteoblasts in vitro and in vivo, showing excellent bone neo-tissue formation.
Materials and Methods: Electrospinning, was used to manufacture porous matrices. Solutions of polylactic acid and two different concentrations of hydroxyapatite, 18wt% and 36wt% were electrospunned. Samples were characterized by scanning electron microscopy (SEM) and probed as a support for cell multiplication of mesenchymal stem cells (MSC´s) differentiated into osteoblasts. Cells were obtained by a biopsy of rabbit’s femur marrow. The differentiation evaluation of MSC´s was performed using antibodies and flow cytometry, also by observing alkaline phosphatase in cells. To further induce cell adhesion and stimulation, a thin layer of polypyrrole doped with iodine (PPy-I), by plasma polymerization, was deposited in the matrices. Samples of PLA/HA/PPy-I underwent in vitro cell culture. Culture was maintained in vitro for 7, 14, 21, and 28 days. At day 7, cells were implanted in vivo, in the back of a rabbit (subcutaneous) for 30 days. Their cell viability was characterized by MTT cell proliferation assay in both cultures.
Results: Fibrous porous matrices were obtained of PLA/HA and PLA/HA/PPy-I fiber diameters varied from 800 nm up to 50 µm and pore size from 10 µm to 100 µm which allows passage of osteoblasts and culture medium within the matrices, as the diameter of osteoblasts is 10 μm. The differentiation of mesenchymal stem cells into osteogenic lineage was confirmed. Matrices showed good cell adhesion and proliferation in vitro and in vivo culture.
Discussion: In the matrices of interest with osteoblasts at day 7 of culture, we can see the beginning of the adhesion of these bone cells.
Figure 1. a) SEM of scaffolds at 7 day of in vitro culture of osteoblasts. a) PLA/HA(18 wt%),
b) PLA/HA(18 wt%)/PPy-I, c) PLA/HA(36 wt%) , d) PLA/HA(36 wt%)/PPy-I
Such cells tend to be in the hydroxyapatite rich areas, adherence is enhanced in the matrices with PPy-I. Also, samples were compared at 7, 14, 21, and 28 days, and, in this case MTT assay showed that samples with PPy-I and 36wt% of hydroxyapatite presented higher cell viability.
Figure 2. Results of MTT assay
When implanted in vivo samples were implanted for 30 days and there was no rejection of the material by the animal, there was vascularization and tissue formation in the matrix, indicating that the material effectively allowed the adhesion and proliferation of cells.
Conclusion: This study confirms that the technique of electrospinning produces porous matrices of PLA/HA. Neotissue is achieved using PLA/HA and PLA/HA/Ppy-I. Better viability was observed when Ppy-I was added to the scaffolds. Increasing the concentration of HA further improved cell viability.
The authors thank the support to CONACYT (Instituto Nacional de Ciencia y Tecnología) for the scholarship.
References:
[1] Tissue engineering of bone: material and matrix considerations. Khan Y., Yaszemski MJ., Mikos AG., et. al. 2008, J Bone Joint Surg Am, pp. 90:36-42.
[2] Biocomposites containing natural polymers and hydroxyapatite for bone tissue. Swetha M., Sahithi K., Moorthi A., et. al. 2010, International Journal of Biological Macromolecules, pp. Volume 47, Issue 1, 1 July 2010, Pages 1–4.
[3] Bio-Hybrid Scaffolds for Bone Tissue Engineering: Nano-Hydroxyapatite/Chitosan Composites. Palazzo B., Izzo D., Scalera F., et. al. 2014, Key Engineering Materials, pp. Bioceramics Volume 26: pages 300-305.
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[5] Plasma polypyrrole implants recover motor function in rats after spinal cord transection. Cruz GJ., Olayo R., Mondragón-Lozano R., et.al. s.l. : Journal of Materials Science: Materials in Medicine , 2012, Vols. Volume 23, Issue 10, pp 2583-2592.