Tetracalcium Phosphate Biocement Hardened with a Mixture of Phytic Acid-Phytase in the Healing Process of Osteochondral Defects in Sheep.

Hyaline articular cartilage has unique physiological, biological, and biomechanical properties with very limited self-healing ability, which makes the process of cartilage regeneration extremely difficult. Therefore, research is currently focused on finding new and potentially better treatment options. The main objective of this in vivo study was to evaluate a novel biocement CX consisting of tetracalcium phosphate-monetit biocement hardened with a phytic acid-phytase mixture for the regeneration of osteochondral defects in sheep. The results were compared with tetracalcium phosphate-monetit biocement with classic fast-setting cement systems and untreated defects. After 6 months, the animals were sacrificed, and the samples were evaluated using macroscopic and histologic methods as well as X-ray, CT, and MR-imaging techniques. In contrast to the formation of fibrous or fibrocartilaginous tissue on the untreated side, treatment with biocements resulted in the formation of tissue with a dominant hyaline cartilage structure, although fine fibres were present (p < 0.001). There were no signs of pathomorphological changes or inflammation. Continuous formation of subchondral bone and hyaline cartilage layers was present even though residual biocement was observed in the trabecular bone. We consider biocement CX to be highly biocompatible and suitable for the treatment of osteochondral defects.

Long-Bone-Regeneration Process in a Sheep Animal Model, Using Hydroxyapatite Ceramics Prepared by Tape-Casting Method.

This study was designed to investigate the effects of hydroxyapatite (HA) ceramic implants (HA cylinders, perforated HA plates, and nonperforated HA plates) on the healing of bone defects, addressing biocompatibility, biodegradability, osteoconductivity, osteoinductivity, and osteointegration with the surrounding bone tissue. The HA ceramic implants were prepared using the tape-casting method, which allows for shape variation in samples after packing HA paste into 3D-printed plastic forms. In vitro, the distribution and morphology of the MC3T3E1 cells grown on the test discs for 2 and 9 days were visualised with a fluorescent live/dead staining assay. The growth of the cell population was clearly visible on the entire ceramic surfaces and very good osteoblastic cell adhesion and proliferation was observed, with no dead cells detected. A sheep animal model was used to perform in vivo experiments with bone defects created on the metatarsal bones, where histological and immunohistochemical tissue analysis as well as X-ray and CT images were applied. After 6 months, all implants showed excellent biocompatibility with the surrounding bone tissue with no observed signs of inflammatory reaction. The histomorphological findings revealed bone growth immediately over and around the implants, indicating the excellent osteoconductivity of the HA ceramic implants. A number of islands of bone tissue were observed towards the centres of the HA cylinders. The highest degree of biodegradation, bioresorption, and new bone formation was observed in the group in which perforated HA plates were applied. The results of this study suggest that HA cylinders and HA plates may provide a promising material for the functional long-bone-defect reconstruction and further research.

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

This study aimed to clarify the therapeutic effect and regenerative potential of the novel, amino acids-enriched acellular biocement (CAL) based on calcium phosphate on osteochondral defects in sheep. Eighteen sheep were divided into three groups, the treated group (osteochondral defects filled with a CAL biomaterial), the treated group with a biocement without amino acids (C cement), and the untreated group (spontaneous healing). Cartilages of all three groups were compared with natural cartilage (negative control). After six months, sheep were evaluated by gross appearance, histological staining, immunohistochemical staining, histological scores, X-ray, micro-CT, and MRI. Treatment of osteochondral defects by CAL resulted in efficient articular cartilage regeneration, with a predominant structural and histological characteristic of hyaline cartilage, contrary to fibrocartilage, fibrous tissue or disordered mixed tissue on untreated defect (p < 0.001, modified O'Driscoll score). MRI results of treated defects showed well-integrated and regenerated cartilage with similar signal intensity, regularity of the articular surface, and cartilage thickness with respect to adjacent native cartilage. We have demonstrated that the use of new biocement represents an effective solution for the successful treatment of osteochondral defects in a sheep animal model, can induce an endogenous regeneration of cartilage in situ, and provides several benefits for the design of future therapies supporting osteochondral defect healing.

Postnatal development of nitrergic and cholinergic structures in rat spinal cord.

Nitric oxide (NO) is known to be a freely diffusible gaseous neurotransmitter that is not requiring synaptic connection to exert its effects. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis can be visualised by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Other neurotransmitter is a classical neurotransmitter acetylcholine (ACh), regulated by enzyme acetylcholinesterase (AChE) that hydrolyses the acetylcholine after its releasing. This work is presenting results of histochemical study of the NADPH-d and AChE expression (nitrergic and cholinergic neurons) in the spinal cord (SC) during various periods in its development. Specimens from Wistar rat pups in the age ranging from 1st to 21st postnatal days (P1-P21) have been compared with those of adult rats (P90). Transverse sections of the SC were evaluated by light microscope. In adults, the NADPH-d positivity was detectable in the neurons of superficial and deep layers of the dorsal horn, pericentral area and in the area of preganglionic autonomic nuclei. AChE positive structures were seen in the same locations as previous ones with the exception of two locations: in superficial layers of the dorsal horn AChE staining was absent, while in the ventral horn the groups of AChE positive motoneurons were found. At the perinatal period both NADPH-d and AChE positive neurons were stained from slight to moderate intensity only. During later developmental periods the staining gradually increased and achieved adult level of intensity on the day P21. Our results confirmed the presence of nitrergic and cholinergic neurons in investigated areas of the SC and indicated their fully functioning of NADPH-d and AChE positive structures in SC from the third postnatal week.

Impact of 2 doses of clorgyline on the rat preimplantation embryo development and the monoamine levels in urine.

We have evaluated the impact of chronic administration of clorgyline, a potent monoamine oxidase A inhibitor and a former antidepressant, on the preimplantation embryo development in Wistar rats. Females were injected intraperitoneally daily for 30 days with saline (control animals), or with a low-dose clorgyline (LDC, 0.1 mg/kg per d) or with a high-dose clorgyline (HDC, 1 mg/kg per d). Embryos were isolated on day 5 of pregnancy and urine was collected by puncture of the urinary bladder. The number of embryos per female did not differ between experimental groups and control, but we have recorded a decreased number of embryos in HDC group compared to LDC (P < .05). We have found that LDC significantly reduced the presence of healthy embryos and increased the presence of the degenerated embryos (P < .001). The administration of the LDC resulted in the lowest cell number in blastocysts. We have observed significantly increased serotonin levels in HDC group compared to both control (P < .05) and LDC animals (P < .01). Norepinephrine (NE) levels in both experimental groups were significantly elevated compared to controls. Dopamine levels did not differ between groups (P > .05). We speculate that lesser negative effect of HDC compared to LDC on the preimplantation embryo development could be the consequence of the lower NE levels and/or elevated serotonin levels. Potential mechanisms mediating clorgyline-induced impaired preimplantation embryo development are proposed.