de Melo Pereira Daniel, Schumacher Matthias, Habibovic Pamela
Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, Netherlands.
Biomater Res. 2021 Mar 20;25(1):6. doi: 10.1186/s40824-021-00209-7.
Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co), which have been previously shown to stimulate resorptive activity of osteoclasts.
To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium.
The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co, which was released in very low amounts, cannot be fully excluded.
This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.
生物矿化胶原蛋白由嵌入羟基磷灰石矿物质的I型纤维状胶原蛋白组成，是一种具有骨替代物潜在应用价值的骨模拟材料。尽管其与骨细胞外基质在化学和结构上相似，但目前尚无证据表明生物矿化胶原蛋白能被破骨细胞吸收。本研究的目的是通过双重修饰诱导破骨细胞对生物矿化胶原蛋白的吸收：增加磷酸钙含量并引入钴离子（Co），先前已证明钴离子可刺激破骨细胞的吸收活性。
为此，我们制备了生物矿化胶原膜并用含钴磷酸钙（CoCaP）进行包被。从外周血CD14 +单核细胞衍生而来的人破骨细胞直接在膜上分化。在对细胞核、F -肌动蛋白和抗酒石酸碱性磷酸酶进行荧光染色后，通过激光共聚焦显微镜对细胞进行分析。通过扫描电子显微镜（SEM）评估其吸收能力，并通过测量钙离子释放到细胞培养基中的量进行间接定量。
CoCaP包被使膜的矿物质含量增加了4 wt.%，弹性模量从1 MPa提高到10 MPa。包被后的膜在水中显示出持续的钴释放，每2天约为7 nM。与未包被的膜相比，在CoCaP包被的生物矿化胶原膜上，破骨细胞偶尔形成肌动蛋白环，并诱导形成吸收陷窝，这通过SEM观察到，并通过细胞培养基中钙浓度的增加得到证实。CoCaP层对破骨细胞功能的影响被认为主要是由膜硬度的增加引起的，尽管释放量非常低的钴的影响不能完全排除。
这项工作表明了这种相对简单的方法诱导破骨细胞对生物矿化胶原蛋白吸收的潜力，尽管破骨细胞吸收的程度有限，且该方法需要进一步优化。此外，这种包被方法适用于将感兴趣的生物活性离子掺入生物矿化胶原蛋白中，而这通常是使用常见的生物矿化方法（如聚合物诱导液体前驱体法）无法实现的。
