Domestic injectable calcium phosphate bone cements for onco-orthopedics: development and biological evaluation

The creation of injectable bone cements (BC) remains a pressing issue in modern biomaterial science due to the prospects for their use in minimally invasive surgical interventions in onco-orthopedics for replacing bone defects in patients that occur after removal of primary or metastatic spinal tumors. An injection of the plastic BC mass into the bone defect of the vertebral body ideally ensures its tight fit and strong cohesion and, later, consolidation with the surrounding tissues, which reduces pain and prevents from the development of compression fractures in patients. In addition to the classic characteristics of osteoplasty materials, namely, cyto-, biocompatibility, osteoconductive potentials, sufficient mechanical strength, some special requirements are imposed on injectable forms of BC, which include the following: viscosity, low thermal effect during mixing, physiological pH value at the implantation site and a certain time span from mixing to solidification, not exceeding 10-15 minutes.

The foreign brands of BC currently available on the biomaterials’ market, along with obvious favorable physical, chemical and biological properties, also have some disadvantages, such as a hyperthermic effect when preparing mixing, acidosis at the implantation site, and not always the optimal time required for solidification.

Over the past few years, the IMET RAS has been developing technologies for the creation of calcium phosphate BC (CPBC), designed, among other things, for vertebroplasty in cancer patients. Their distinctive feature is the absence of a thermal effect during mixing, pH values close to physiological in the defect zone after implantation, and the optimal time of solidification

The aim of this study is a biological assessment in vitro of CPBC samples developed by the IMET RAS.

MATERIALS AND METHODS

The synthesis of cement powders was carried out in accordance with the previously described approach (Goldberg et al., 2020). For the purpose of biological assessment, cement powders based on the magne-sium-substituted whitlockite phase were used, characterized by the ratio (Ca + Mg) / P = 1.67 and the replacement of calcium with magnesium by 20 mol.%, without grinding and mechanically activated by grinding to create a homogeneous cement paste. The CPBC samples were prepared by mixing the initial components (cement powder and liquid (L)). Two compositions were used as L: 1 - an aqueous solution based on 50 mass % NaH2PO4 (L-1), 2 – liquid L-1 was mixed with 0.75 mass % aqueous solution of carboxymethyl cellulose (CMC) at a ratio of 1: 1 (L-2). Thus, the in vitro study was performed on 4 samples of CPBC, among which 2 samples (No. 1 and 3) were obtained from cement powders without grinding using L-1 and L-2, respectively, and 2 samples (No. 2 and 4) based on mechanically activated powders both with L-1 and L-2.

Our in vitro studies to assess the acute toxicity and cytocompatibility of CPBC samples were performed on the model of the transplantable human osteosarcoma cell line MG-63 (Russian Collection of Vertebrate Cell Cultures, Institute of Cytology, Russian Academy of Sciences, St. Petersburg).

The cytotoxicity of CPBC samples was studied according to GOST R ISO 10993 (parts 5 and 12), by culturing MG-63 cells in the presence of extracts of the above material samples (indirect contact). The addition of extracts (extracts) from the samples to the cells was carried out 24 hours after seeding the cells in the wells in the state of a subconfluent monolayer of the MG-63 culture. As a negative reference, a complete growth medium (CGM) was added to the cells, and as a positive reference used was a 50% solution of dimethyl sulfoxide (DMSO, PanEco, Russia) in CGM. The cultivation period of MG-63 cells with extracts of the CPBC samples was 24 hours.

Studies to assess the cytocompatibility of the CPBC samples were carried out by direct contact of the MG-63 culture with the surface of the studied material samples. Cultivation was supported for 1, 3, 7 days with regular (twice a week) change of medium. Wells with cells on polystyrene culture plastic served as a reference. Cell viability at the cultivation stages was determined using the MTT test (T. Mossman, 1983). The optical density of the formazan solution (reaction product) was estimated using the Multiscan FC spectrophotometer (Thermoscientific, USA) at a wavelength of 540 nm.

For each studied sample of CPBC at a specific cultivation period, the population of viable cells (PVC) in the experiment was calculated in relation to the reference (in %) using the formula as follows: PVC = OD_experiment: OD_reference x 100%, where OD is the optical density of the formazan solution in the experiment and in the negative reference, respectively. Next, when assessing the toxicity of the samples, the toxicity index (TI) of the material samples was calculated using the formula as given below: TI = 100% – OD_experiment / OD reference x 100%.

A material sample was considered non-toxic with its TI value ≤30% and cytocompatible with a PVC value ≥70% at the stages of the experiment. In addition, when studying cytocompatibility at the end of cultivation, the value of the increase in the cell population in the reference and for each experimental sample of materials (in percent) was calculated as the ratio of the difference in the OD value of the formazan solution on cultivation day 7 cultivation day 1 to the OD value of the formazan solution on observation day 1.

RESULTS

It was shown that the pH value of the extracts from the developed samples of the CPBC depends on the composition of the L: it is in the physiological range (7.14-7.3) for the samples of cements mixed with L-1 and in the slightly alkaline range (7.8-7.9) for the cements mixed with L-2.

The given samples of cements do not demonstrate cytotoxicity: after 24 hours of culturing human osteo- 28 | Cardiometry | Issue 33. November 2024

sarcoma cells MG-63 in the presence of their extracts, the viability of the test culture remained at a level of 70.2 - 95.8% (see Table 1 herein).

Table 1

The value of the optical density of the formazan solution (OD, MTT test), the pool of viable cells (PVC) and the toxicity index (TI) during the cultivation of human osteosarcoma cells MG-63 with extracts of samples of CPBC No. 1-4 (24 hours of cultivation)

*Significant difference from the reference according to the Student’s criterion (p<0.05)

Upon direct contact of the test culture with the cement samples, MG-63 cells attached to their surface and began to actively proliferate in the period from the 1st to the 4th day of cell growth at a rate comparable to the reference (cells on polystyrene) and later, due to the cell culture reaching the level of confluence, reached a plateau. The value of the cell population increase per week of culturing MG-63 cells on the studied CPBC samples was 244-350% vs. 280% in the reference. At the same time, the rate of cell expansion of the CPBC samples did not depend on the composition of the L and the methods of preparing the cement powders (without grinding or mechanically activated) (see Table 2 herein). The obtained results bear witness to the cytocompatibili-ty of all 4 developed CPBC samples.

CONCLUSION

Compositions of domestic injectable CPBCs for onco-orthopedics have been developed. The first stage of their biological evaluation has been carried out as follows: the in vitro experiments have proven the absence of cytotoxicity of these material samples and demonstrated their cytocompatibility. After their further in vivo study on a model of bone defect in laboratory animals, the most promising ones will be selected to complete the preclinical stage of their testing.

Table 2

The value of the optical density in the formazan solution (OD, MTT test), PVC and population growth of the human osteosarcoma line MG-63 during cultivation on polystyrene (reference) and CPBC samples (experiment) in the observational dynamics