## 3 Hydroxyapatite Particles in Scaffold Manufacturing and Cell Signaling
### Antimicrobial Scaffolds  
As previously mentioned, there is one more aspect to be taken into account by scientists in order to overcome the infections that might appear at the defect site--finding an antimicrobial mixture which can reduce the bacterial adhesion and biofilm formation [56; 57]. Until now, it was discovered that some materials can inhibit bacterial attachment both in vitro and in vivo [58; 59]. For example, in a study conducted by [PERSON] in 2020, researchers developed chitosan-silk fibroin-based materials in multiple combinations: the first set of samples was made with polyethylene terephthalate, the second set with polyethylene terephthalate and HA, and the third set with polyethylene terephthalate and HA-silver nanoparticles. All materials were obtained by using the plasma-splash technique. These setsof samples were subjected to in vitro and in vivo testing in rabbits. It was concluded that the best result in terms of cellular attachment was observed in the case of scaffolds with HA-silver in contact with mesenchymal stem cells. Moreover, osteoinduction was achieved by the HA-silver, a fact confirmed by evaluation after 14 days in contact with the same cellular type, when upregulation of multiple growth factors such as VEGF (promotes angiogenesis) and osteoclastin was validated [60]. Additionally, [PERSON] et al. proved that alginate could be used as a coating for porous HA scaffolds with the aim of immobilizing biomolecules and drugs (dexamethasone and stromal cell factor 1) in the form of microspheres. In vitro cell culture tests and in vivo evaluation on dogs confirmed multiple expected theories such as that: stromal cell factor 1 had an influence on cellular signaling and accelerated the recruitment of mesenchymal stem cells, the release of dexamethasone directed the differentiation of the same cellular line to osteogenic phenotype, and the results collected from canine models showed that newly-formed bone tissue was observed in the depth of the porous structure of the scaffolds [61]. Proper results were obtained in the case of human fetal osteoblasts as well by [PERSON] et al., who used polycaprolactone/zinc oxide scaffolds nanostructures loaded with HA particles. The choice of zinc oxide might be explained due to its corrosion rate which is very close to the one possessed by the natural bone tissue and because it is a fundamental element for the good functioning of a living human organism, being essential for the normal activity of about 3000 proteins. Moreover, it showed good antimicrobial properties and it acts as an osteoinductive stimulator [62]. The results for this type of scaffold showed that the ALP (Alkalaine phosphatase) activity in contact with the above-mentioned cell phenotype was significantly higher--114 times more cellular viability was observed in the case of scaffolds containing HA and 6% zinc oxide when compared to simple polycaprolactone scaffolds [1; 63]. Carbon dots were also investigated in combination with nanostructured HA and chitosan as scaffolds to evaluate the impact of carbon dots on ossification, reduction of bacterial attachment, and in treating osteosarcoma by photothermal treatment. In vitro studies on bone mesenchymal stem cells isolated from rats concluded that the addition of carbon dots and nanostructured HA is able to guide stem cell osteogenesis and a higher expression of ALP in comparison with simple chitosan-nanostructured HA scaffolds. After 2 weeks, collagen I and osteoclastin expression were higher for scaffolds containing carbon dots. In terms of their compatibility for tumor therapy, carbon dots exhibited photothermal effects in the presence of near-infrared spectrum radiations. Moreover, when subjected to this type of radiation their antimicrobial performance was present and the rate of _S. aureus_ inhibition was about 99%, where for _E. coli_, the inhibition rate was 97% [64]. While antimicrobial properties do not play a direct role in the process of bone regeneration, prevention of infections at the damaged site is crucial.