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Porous polyhydroxybutyrate (PHB)-chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends.

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(1) Background: The preparation and characterization of novel fully injectable enzymatically hardened tetracalcium phosphate/monetite cements (CXI cements) using phytic acid/phytase (PHYT/F3P) hardening liquid with a small addition of polyacrylic acid/carboxymethyl cellulose anionic polyelectrolyte (PAA/CMC) and enhanced bioactivity. (2) Methods: Composite cements were prepared by mixing of calcium phosphate powder mixture with hardening liquid containing anionic polyelectrolyte. Phase and microstructural analysis, compressive strength, release of ions and in vitro testing were used for the evaluation of cement properties. (3) Results: The simple possibility to control the setting time of self-setting CXI cements was shown (7–28 min) by the change in P/L ratio or PHYT/F3P reaction time. The wet compressive strength of cements (up to 15 MPa) was close to cancellous bone. The increase in PAA content to 1 wt% caused refinement and change in the morphology of hydroxyapatite particles. Cement pastes had a high resistance to wash-out in a short time after cement mixing. The noncytotoxic character of CX cement extracts was verified. Moreover, PHYT supported the formation of Ca deposits, and the additional synergistic effect of PAA and CMC on enhanced ALP activity was found, along with the strong up-regulation of osteogenic gene expressions for osteopontin, osteocalcin and IGF1 growth factor evaluated by the RT-qPCR analysis in osteogenic αMEM 50% CXI extracts. (4) Conclusions: The fully injectable composite calcium phosphate bicements with anionic polyelectrolyte addition showed good mechanical and physico-chemical properties and enhanced osteogenic bioactivity which is a promising assumption for their application in bone defect regeneration.

Novel calcium phosphate cements containing a mixture of four amino acids, glycine, proline, hydroxyproline and either lysine or arginine (CAL, CAK) were characterized and used for treatment of artificial osteochondral defects in knee. It was hypothesized that an enhanced concentration of extracellular collagen amino acids (in complex mixture), in connection with bone cement in defect sites, would support the healing of osteochondral defects with successful formation of hyaline cartilage and subchondral bone. Calcium phosphate cement mixtures were prepared by in situ reaction in a planetary ball mill at aseptic conditions and characterized. It was verified that about 30–60% of amino acids remained adsorbed on hydroxyapatite particles in cements and the addition of amino acids caused around 60% reduction in compressive strength and refinement of hydroxyapatite particles in their microstructure. The significant over-expression of osteogenic genes after the culture of osteoblasts was demonstrated in the cement extracts containing lysine and compared with other cements. The cement pastes were inserted into artificial osteochondral defects in the medial femoral condyle of pigs and, after 3 months post-surgery, tissues were analyzed macroscopically, histologically, immunohistochemically using MRI and X-ray methods. Analysis clearly showed the excellent healing process of artificial osteochondral defects in pigs after treatment with CAL and CAK cements without any inflammation, as well as formation of subchondral bone and hyaline cartilage morphologically and structurally identical to the original tissues. Good integration of the hyaline neocartilage with the surrounding tissue, as well as perfect interconnection between the neocartilage and new subchondral bone tissue, was demonstrated. Tissues were stable after 12 months’ healing.

Polyhydroxybutyrate/chitosan/calcium phosphate composites are interesting biomaterials for utilization in regenerative medicine and they may by applied in reconstruction of deeper subchondral defects. Insufficient informations were found in recent papers about the influence of lysozyme degradation of chitosan in calcium phosphate/chitosan based composites on in vitro cytotoxicity and proliferation activity of osteoblasts. The effect of enzymatic chitosan degradation on osteoblasts proliferation was studied on composite films in which the porosity of origin 3D scaffolds was eliminated and the surface texture was modified. The significantly enhanced proliferation activity with faster population growth of osteoblasts were found on enzymatically degraded biopolymer composite films with α-tricalcium phosphate and nanohydroxyapatite. No cytotoxicity of composite films prepared from lysozyme degraded scaffolds containing a large fraction of low molecular weight chitosans (LMWC), was revealed after 10 days of cultivation. Contrary to above in the higher cytotoxicity origin untreated nanohydroxyapatite films and porous composite scaffolds. The results showed that the synergistic effect of surface distribution, morphology of nanohydroxyapatite particles, microtopography and the presence of LMWC due to chitosan degradation in composite films were responsible for compensation of the cytotoxicity of nanohydroxyapatite composite films or porous composite scaffolds.

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