An adverse effect of this fast degr

CS支架的这种快速降解速率的不利影响是培养基的pH值显着增加。如果提取物未稀释至低于一定浓度（例如，本研究中为0.02 g / mL），则会抑制细胞生长。当生物陶瓷材料具有相对更快的降解速率时，这是一种常见现象[59,60]。在报告中，如果主要的生物活性成分来自陶瓷支架的降解，则通常也要在稀释的提取物中进行体外细胞培养[61-63]。例如，Bun-petch等人。用含硅CaPs提取物以不同浓度（1.56–25 mM）培养BMSCs时，能够促进细胞增殖和成骨分化的最佳浓度为6.25 mM [61]。在另一份报告中，遵循ISO / EN 10993-5标准[62]，将由β-CS制成的稀释提取物用于培养成骨细胞样细胞。结果表明，提取物的浓度高于50 mg / mL时，对细胞增殖具有抑制作用。然而，在本研究（图8）和文献[4,38,64]中，这些生物陶瓷支架都可以支持细胞附着，并且接种的细胞表现出强大的细胞活力。同样，这些支架可以在动物评估中很好地发挥作用，而不会在植入部位和其他器官（如肝和肾）引起明显的炎症反应（图11和图S10）。没有确切的解释可以说明浓缩提取物中细胞培养与接触模式下培养细胞的不一致。细胞与系统相互作用的动力学可能不同。在细胞培养期间，细胞连续暴露于预先准备的浓缩提取液中，而接种在支架上的细胞则暴露于动态离子浓度下，同时逐渐释放离子并恢复培养基。当这些支架体内植入时，它们与周围组织/细胞的相互作用也处于动态模式。结果，与空白对照相比，当植入任一CS支架（CS-g，10Mg-CS-g，10Mn-CS-g）时，大鼠颅骨缺损均得到了明显的再生。同时将接种在支架上的细胞暴露于动态离子浓度下，并逐渐释放离子并恢复培养基。当这些支架体内植入时，它们与周围组织/细胞的相互作用也处于动态模式。结果，与空白对照相比，当植入任一CS支架（CS-g，10Mg-CS-g，10Mn-CS-g）时，大鼠颅骨缺损均得到了明显的再生。同时将接种在支架上的细胞暴露于动态离子浓度下，并逐渐释放离子并恢复培养基。当这些支架体内植入时，它们与周围组织/细胞的相互作用也处于动态模式。结果，与空白对照相比，当植入任一CS支架（CS-g，10Mg-CS-g，10Mn-CS-g）时，大鼠颅骨缺损均得到了明显的再生。

CS 支架的这种快速降解率的一个负面影响是介质的 pH 值显著增加。如果提取物不稀释到低于特定浓度（例如本研究中0.02克/mL），这些提取物会抑制细胞生长。这是与生物抗癌材料相关的常见现象，因为它们的降解率相对较快[59，60]。在报告中，体外细胞培养通常也进行稀释提取物，如果主要生物活性成分来自陶瓷scffold的降解[61[63]。例如，Bun-petch等人用不同浓度（1.56~25 mM）的含有Si的CaPs的提取物培养BMSC，发现能够促进细胞增殖和骨质分化的最佳浓度为6.25 mM[61]。在另一份报告中，β-CS的稀释提取物在ISO/EN 10993-5标准[62]的指导下用于孵育成骨细胞。结果表明，该提取物浓度高于50mg/mL，对细胞增殖有抑制作用。然而，本研究（图8）和文献[4，38，64]，这些生物脑支架可以支持细胞附着，种子细胞表现出强大的细胞生存能力。此外，这些类型的脚手架可以在动物评估中很好地作用，而不会在植入点和其他器官（如肝脏和肾脏）中引起明显的炎症反应（图11和图）。S10）。没有确切的解释来阐明在浓缩提取物和接触模式下培养的细胞培养的不协调性。细胞与系统相互作用可能归因于不同的动态。细胞在细胞培养过程中不断暴露在预先准备的浓缩提取物中，而脚手架上播种的细胞则暴露在动态离子浓度中，同时伴随着逐渐的离子释放和中等的茶点。当这些脚手架被植入体内时，它们与周围组织/细胞的相互作用也处于动态模式。结果，在植入其中任何一个CS支架（CS-g，10Mg-CS-g，10Mn-CS-g）时，与空白控制相比，大鼠的卡路里缺陷显著再生。

An adverse effect of this fast degradation rate of CS scaffolds was the remarkable increase in the pH values of the media. The extracts would inhibit cell growth if they were not diluted to below certain concentrations (e.g. 0.02 g/mL in this study). This is a common phenomenon in relation to bioceramic materials when they have relative faster degradation rates [59,60]. In reports, in vitro cell culture was also normally carried out in diluted extracts if the main bioactive components come from the degradation of ceramic scffolds [61–63]. For example, Bun-petch et al. cultured BMSCs with the extracts made from Si-containing CaPs at different concentrations (1.56–25 mM), the optimal concentration found able to promote cell proliferation and osteogenic differentiation was 6.25 mM [61]. In another report, diluted extracts made from β-CS were used to incubate osteoblast-like cells following the guidance of ISO/EN 10993-5 standard [62]. The results showed that the extract had an inhibitory effect on cell proliferation as its concentration being higher than 50 mg/mL. Both in the present study (Fig. 8) and in literature [4,38,64], however, these bioceramic scaffolds could support cell attachment, and the seeded cells demonstrated strong cell viability. Also, these kinds of scaffolds could act well in animal evaluations without causing apparent inflammation reactions both in the implantation sites and to other organs like liver and kidney (Fig. 11 and Fig. S10). There was no exact explanation to illuminate the inconsis- tency of the cell culture in the concentrated extracts and the cells cultured in the contact mode. It might be ascribed to the different dy- namic that cells interacted with the systems. The cells were continuously exposed to the pre-prepared concentrated extracts during the cell cul- ture, while the cells seeded on the scaffolds were exposed to dynamic ion concentrations alongside gradual ion release and medium refreshment. When these scaffolds were in vivo implanted, their interactions with surrounding tissues/cells were also in a dynamical mode. As the results, the rat calvarial defects were regenerated significantly in comparison with the blank control when either of the CS scaffolds (CS-g, 10Mg-CS-g, 10Mn-CS-g) was implanted.<br>