Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, Warsaw 02-507, Poland.
Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, Warsaw 02-507, Poland.
Biomater Adv. 2023 Jun;149:213403. doi: 10.1016/j.bioadv.2023.213403. Epub 2023 Mar 24.
Biomimetic production of coatings on various types of scaffolds is based mainly on simulated body fluid precipitation (SBF) of apatites, or, if the HCO is present, carbonated apatites. Recently, we proposed formation of calcium phosphates (CaP) precipitates by alkaline phosphatase (ALP) hydrolysing glycerophosphate in presence of calcium ions as an alternative to SBF. Since apatites synthesized in bone by the ALP activity contain carbonate anions, it was tempting to investigate whether the phosphatase method could be advanced into osteomimetic one. Therefore, taking example from the SBF studies, phosphatase incubation medium was enriched with carbonate ions at 4.2 and 27 mM concentration. X-ray diffraction of the precipitates disclosed peaks typical for hydroxyapatite (HAP). FTIR analysis showed that at both concentration of carbonate ions, apatites underwent both B and A substitution, more extensive at higher concentration. Thus, osteomimetic approach produced carbonated hydroxyapatites of the type encountered in bone tissue even at HCO concentration as low as 4.2 mM. Composite plates made of poly(ε-caprolactone) and mixture of β-tricalcium phosphate and hydroxyapatite at mass ratio of 1:0.5:0.5, respectively, were covered by CaP coatings, i.e., CaP-0, CaP-4.2, CaP-27, by incubation in phosphatase medium containing 0, 4.2 or 27 mM of NaHCO, respectively. Pristine or coated PCL50 plates were used to study release of calcium and adsorption/desorption of proteins, or seeded with human bone marrow mesenchymal stem cells (hMSC) for study of cell adhesion, spreading and osteogenic differentiation. Introduction of carbonate into the CaP coatings significantly increased release of Ca in a carbonate concentration-dependent manner; the release was up to 4 times higher, when compared to CaP-0 coating, and reached 0.41 ± 0.01 mM for CaP-27 after first 24 h. Coating CaP-4.2 yielded significantly higher adsorption of bovine serum albumin and cytochrome C than CaP-0. All of the CaP coatings improved significantly hMSC adhesion, however, only CaP-4.2 provided 2 times higher cell number than PCL50 after 2 weeks of culture. Interestingly, ALP activity calculated per cell number was the highest on pristine plates, presumably because hMSC differentiate preferentially into osteoblasts at lower seeding densities. It appears, therefore, that the osteomimetic approach may be useful for production of carbonated hydroxyapatite coatings, but requires further studies and replacing intestinal phosphatase used in this work with one originating from bone.
仿生法在各种类型的支架上生成涂层主要基于模拟体液沉淀（SBF）生成磷灰石，或者，如果存在 HCO，生成碳酸磷灰石。最近，我们提出了一种替代 SBF 的方法，即通过碱性磷酸酶（ALP）水解甘油磷酸，在钙离子存在的情况下生成磷酸钙（CaP）沉淀物。由于 ALP 活性在骨中合成的磷灰石含有碳酸根阴离子，因此我们试图研究这种磷酸酶方法是否可以进一步发展为骨仿生方法。因此，受 SBF 研究的启发，在含有 4.2 和 27mM 浓度碳酸根离子的磷酸酶孵育介质中合成 CaP 沉淀物。沉淀物的 X 射线衍射显示出羟磷灰石（HAP）的典型峰。傅里叶变换红外分析表明，在两种碳酸根离子浓度下，磷灰石都经历了 B 和 A 取代，高浓度下的取代更为广泛。因此，即使在 HCO 浓度低至 4.2mM 的情况下，骨仿生方法也能生成在骨组织中遇到的碳酸羟基磷灰石。由聚（ε-己内酯）和β-磷酸三钙与羟磷灰石的质量比为 1:0.5:0.5 的混合物制成的复合板分别用在含有 0、4.2 或 27mM NaHCO 的磷酸酶孵育液中覆盖 CaP 涂层，即 CaP-0、CaP-4.2、CaP-27。用未处理或涂覆的 PCL50 板研究钙的释放和蛋白质的吸附/解吸，或接种人骨髓间充质干细胞（hMSC）研究细胞黏附、铺展和成骨分化。将碳酸根引入 CaP 涂层中，以碳酸根浓度依赖的方式显著增加了 Ca 的释放；与 CaP-0 涂层相比，释放量增加了 4 倍，在 24 小时后达到 CaP-27 的 0.41±0.01mM。CaP-4.2 涂层对牛血清白蛋白和细胞色素 C 的吸附明显高于 CaP-0。所有的 CaP 涂层都显著提高了 hMSC 的黏附能力，然而，在培养 2 周后，只有 CaP-4.2 提供的细胞数量比 PCL50 高 2 倍。有趣的是，根据细胞数量计算的碱性磷酸酶活性在未处理的平板上最高，这可能是因为 hMSC 在较低的接种密度下优先分化为成骨细胞。因此，骨仿生方法似乎可用于生成碳酸羟基磷灰石涂层，但需要进一步研究，并将本工作中使用的来自肠道的碱性磷酸酶替换为来自骨骼的碱性磷酸酶。
