Characterization of Properties, In Vitro and In Vivo Evaluation of Calcium Phosphate/Amino Acid Cements for Treatment of Osteochondral Defects

Abstract

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.

Tetracalcium Phosphate/Monetite/Calcium Sulfate Hemihdrate Biocement Powder Mixtures Prepared by the One-Step Synthesis for Preparation of Nanocrystalline Hydroxyapatite Biocement-Properties and In Vitro Evaluation

“…Tetracalcium phosphate (Ca 4 (PO 4 ) 2 O, TTCP) was prepared according to the method described in [ 9 ]. The TTCPM/nanoCSH powder cement mixtures were synthesized via an in situ reaction of TTCP with orthophosphoric acid (86% analytical grade, Merck, Darmstadt, Germany)/H 2 SO 4 (96%, analytical grade, Merck) mixture in 80 v / v % ethanol (reaction solution) using a planetary ball mill with agate balls and a vessel for 30 min.…”

“…The gene expression was analyzed using a similar method as that in Reference [ 9 ]. For the extraction of total RNA, we used approximately 1 × 10 6 cells.…”

“…The rise in cement solubility with enhanced release of calcium from the cement improves the bioactivity of cement in relation to osteoinduction [ 8 ]. Another possibility for improvement of TTCPM bioactivity is addition of more soluble inorganic components, which actively influence cell behavior after the application of cements; from this point of view, the addition of a complex amino acid mixture or arginine-glycine-aspartate (RGD) [ 9 , 10 ], the preparation of TTCPM/magnesium phosphate composite cements with more soluble magnesium components [ 11 ], substitution of HAP with sulfate ions [ 12 ], and CS/CPC composites [ 13 ] represent cheap and simple examples of studied biocements. Calcium sulfate in the form of hemihydrate (CSH) has been used as a cheap, synthetic, non-toxic, and bioactive bone filler over 100 years.…”

In Vivo Study of Osteochondral Defect Regeneration Using Innovative Composite Calcium Phosphate Biocement in a Sheep Model

“…Biocements were prepared according to Medvecky et al [ 31 ]. Tetracalcium phosphate Ca 4 (PO 4 ) 2 O was made by annealing process of an equimolar mixture of dicalcium phosphate anhydrous CaHPO 4 (Fluka, Buchs, Switzerland) and calcium carbonate CaCO 3 (Sigma-Aldrich, Saint Louis, MI, USA) for 5 h at the temperature 1450 °C.…”

“…The characteristic and properties of C and CAL cements can be found in our previous article [ 31 ]. In the microstructure of C cement, we found a low number of larger pores (10–15 µm) and a high fraction of 1–3 µm size micropores.…”