## 4 Discussion  
Although CS-CP-based premixed putty has good biocompatibility, osteo-conduction, and osteo-integration, the poor anti-washout ability limits the clinical utility of premixed putty. HPMC and glycerin could increase the cohesion and maintain stability before use [27; 33]. Our results indicated the addition of HPMC could effectively improve the anti-washout properties of premixed putty; as HPMC increased, premixed putty was less washed out. The possible mechanism of HPMC action can be summarized into two aspects. On the one hand, it has been shown that HPMC can provide 3D networking for materials such as mixing with collagen [23; 40], and without HPMC the material structure was looser so it falls apart easily. It is possible that HPMC provided stronger structure for premixed putty, thus we observed that the anti-washout property was better when there was more HPMC added into the premixed putty (Figure 2A). On the other hand, HPMC has a hemostatic effect by water adsorption capacity [40], the water competition between CSH and HPMC. Addition of HPMC was predicted to have a negative effect on CSH hydration, and there was an effect on the hydration reaction of the washed particle. For 2 wt% and 4 wt% HPMC groups, peaks of CSH still existed after soaking in SBF, which can be seen from XRD (Figure 4B). The possible reason for this is that the gel network prevented particles from washing out, but it also makes CSH inaccessible to water [41]. In situ bone substitute containing CS and \(\beta\)-TCP has been clinically studied with good outcomes, and used in osteotomies, vertebral bodies, fracture surgery, and bone tumors [1; 2; 3]. However, the bone substitute is often in direct contact with the muscle and sometimes leaking out from the bone defect area, which may lead to a local inflammatory process at the implantation site [14; 15]. The degree of inflammatory response is related to the number, size and morphology of collapsed particles [42; 43]. Our results indicated that when HPMC percentage was increased, the disintegration of premixed putty can be improved from more than 70% to less than 40% (Figure 2B), resulting in a reduction in the particle numbers (Figure 2B). The inflammatory response is related to the size and shape of the particle; when its size is less than 10 \(\upmu\)m or in a needle-like morphology, it produces a relatively strong inflammatory response [42; 43]. For the putty group with 4 wt% HPMC, it was affected by the proportion of particles whose sizes were lower than 10 \(\upmu\)m, but it had no significant effect on the other groups (Table 3). The possible reason for this is that  
Figure 4: X-ray diffractometry patterns of premixed putty containing HPMC with different mass ratios after being soaked for 4 h (*: \(\beta\)-TCP; \(\diamond\): calcium sulfate dihydrate (CSD), \(\blacksquare\): CSH). (**A**) Patterns of the main body. (**B**) Patterns of particles. the particles were coated with and hydrated between each other with HPMC because it absorbed water molecules on the surface and formed a monomolecular layer, resulting in increased Van der Waals forces and reduced inter-particle separation [41]. However, our results indicated that the morphology of washed particles had no significant effect on the aspect ratio of the particles (Table 3) with irregular and rod-like shapes (Figure 3B). When the size of the particles was smaller than 10 \(\upmu\)m [44], the macrophage tended to respond and the particles were phagocytosed, resulting in inflammation. Our results showed that when the premixed putty contained 4% HPMC, only 6.4% of particles were smaller than 10 \(\upmu\)m, suggesting that less inflammation could occur. When the premixed putty contained 1% and 2% HPMC, the percentage of particles smaller than 10 \(\upmu\)m was similar to the control, thus it may result in inflammation. However, further investigation should be conducted in order to verify this possibility.