Poly(3-hydroxybutyrate)/poly(ethylene glycol) scaffolds with different microstructure: the effect on growth of mesenchymal stem cells

Abstract

Development of biocompatible 3D scaffolds is one of the most important challenges in tissue engineering. In this study, we developed polymer scaffolds of different design and microstructure to study cell growth in them. To obtain scaffolds of various microstructure, e.g., size of pores, we used double- and one-stage leaching methods using porogens with selected size of crystals. A composite of poly(3-hydroxybutyrate) (PHB) with poly(ethylene glycol) (PEG) (PHB/PEG) was used as polymer biomaterial for scaffolds. The morphology of scaffolds was analyzed by scanning electron microscopy; the Young modulus of scaffolds was measured by rheometry. The ability to support growth of mesenchymal stem cells (MSCs) in scaffolds was studied using the XTT assay; the phenotype of MSC was preliminarily confirmed by flow cytometry and the activity of alkaline phosphatase and expression level of CD45 marker was studied to test possible MSC osteogenic differentiation. The obtained scaffolds had different microstructure: the scaffolds with uniform pore size of about 125 µm (normal pores) and 45 µm (small pores) and scaffolds with broadly distributed pores size from about 50-100 µm. It was shown that PHB/PEG scaffolds with uniform pores of normal size did not support MSCs growth probably due to their marked spontaneous osteogenic differentiation in these scaffolds, whereas PHB/PEG scaffolds with diverse pore size promoted stem cells growth that was not accompanied by pronounced differentiation. In scaffolds with small pores (about 45 µm), the growth of MSC was the lowest and cell growth suppression was only partially related to stem cells differentiation. Thus, apparently, the broadly distributed pore size of PHB/PEG scaffolds promoted MSC growth in them, whereas uniform size of scaffold pores stimulated MSC osteogenic differentiation.

Recent developments in the synthesis of poly(hydroxybutyrate) based biocomposites

“…Biocompatible and hydrophilic properties of the composite can be improved with the incorporation of PEG into PHB. Bonartsev et al developed PHB‐PEG composite scaffolds using ammonium carbonate and sucrose as porogens via salt leaching method. PHB‐PEG biocomposite had a bulk density of 1.08 g/cm 3 and Young's modulus in the range from 35 ± 1 to 110 ± 2 kPa.…”

“…PHB has 60–80% degree of crystallinity and stereochemical regularity, which limit its applications in biomedical field . PEG can reduce the crystallinity of PHB as PHB/PEG composite scaffolds has been developed to promote the growth of mesenchymal stem cells (MSCs) of rats . The sutures made of PHB and PHBV containing 15 mol% hydroxyvalerate has been prepared.…”

The Creation of Tissue-Engineered Therapeutic System Based on Hybrid Construction From Poly-3-Oxybuxyrate With Hydroxyapathitis Completed With Alginate Hydrogel and Mesenchimal Stem Cells

“…Для получения скаффолдов был использован ПОБ (ММ=130 кДа), полученный по оригинальной методике контролируемого биосинтеза с использованием штамма-продуцента Azotobacter chroococcum 7Б [11]. Помимо ПОБ в работе были использованы следующие материалы и реактивы: гидроксиапатит (Сигма-Алдрич, Германия), альгинат натрия (Сигма-Алдрич, Германия), МСК выделены из костного мозгах 3-х дневных крысят, трихлорметан (EKOS-1, Russia), карбонат аммония (Химмед, Россия), сахароза (Химмед, Россия), пруток из полилактида диаметром 1,75 мм 3D для печати прессформ методом послойного наплавления (температура плавления 200-255°С, плотность 1,2-1,08 кг/м 2 , FDPlast, Россия), Золетил 100 (Virbac, Франция), доксициклин (Бинергия ЗАО (Россия), тетрациклин (Биохимик ОАО, Россия), ализариновый красный (ХимМедСервис, Россия).…”

“…Для исследования роста МСК на биополимерных композитах и биокомпозитных скаффолдах in vitro были использованы МСК крыс, выделенные из костного мозга 3-х дневных крысят. Клетки культивировали в среде ДМЕМ в стандартных условиях [11]. Все эксперименты были проведены согласно правилам гуманного обращения с лабораторными животными по ГОСТ ISO 10993-1-2011.…”

“…Количество клеток определяли по калибровочной кривой. Затем рассчитывали относительное количество клеток в процентах от начального их количества, добавленного к образцу скаффолда [11].…”