Abstract
Biphasic bone substitutes (BBS) are currently well-established biomaterials. Through their constant development, even natural components like hyaluronic acid (HY) have been added to improve both their handling and also their regenerative properties. However, little knowledge exists regarding the consequences of the addition of HY to their biocompatibility and the inflammatory tissue reactions. Thus, the present study was conducted, aiming to analyze the influence of two different amounts of high molecular weight HY (HMWHY), combined with a BBS, on in vitro biocompatibility and in vivo tissue reaction. Established in vitro procedures, using L929 cells, were used for cytocompatibility analyses under the test conditions of DIN EN:ISO 10993-5. For the in vivo part of the study, calvarial defects were created in 20 Wistar rats and subsequently filled with BBS, and BBS combined with two different HMWHY amounts, i.e., BBS + HY(L) and BBS + HY(H). As controls, empty defects were used. Established histological, immunohistochemical, and histomorphometrical methods were applied to analyze the tissue reactions to the three different materials, including the induction of pro- and anti-inflammatory macrophages and multinucleated giant cells (BMGCs). The in vitro results showed that none of the materials or compositions caused biological damage to the L929 cells and can be considered to be non-toxic. The in vivo results showed that only the addition of high doses of HY to a biphasic bone substitute significantly decreases the occurrence of pro-inflammatory macrophages (* p < 0.05), comparable to the numbers found in the control group, while no significant differences within the three study groups for M2-macrophages nor BMGCs were detected. In conclusion, the addition of different amounts of HMWHY does not seem to affect the inflammation response to BBS, while improving the material handling properties.
Highlights
Bone substitute materials (BSM) are in daily use for different applications in the medical fields of orthopedics, traumatology, and dentistry
A mean of 2.81 ± 1.18 CD163-positive biomaterial-induced multinucleated giant cells (BMGCs)/mm2 were found in the group of the pure bone substitutes (BBS), while in the group of the BBS combined with the low concentration of hyaluronic acid (HY) it was 2.31 ± 1.47 CD163-positive BMCGs/mm2 and in the group of the BBS combined with the high concentration of HY 3.59 ± 2.06 CD163-positive BMGCs/mm2 were measured (Figure 4)
A mean of 5.18 ± 1.56 CD206-positive BMGCs/mm2 were detected in the group of the pure BBS, while in the group of the BBS combined with the low concentration of HY it was 5.85 ± 1.30 CD206-positive BMCGs/mm2 and in the group of the BBS combined with the high concentration of HY 6.47 ± 1.67 CD206-positive BMGCs/mm2 were detected (Figure 4)
Summary
Bone substitute materials (BSM) are in daily use for different applications in the medical fields of orthopedics, traumatology, and dentistry. It has been determined that the mixture of both compounds with a HA/β-TCP ratio of 60/40 wt% induced a tissue reaction including a high BMGC formation and vascularization rate comparable to pure β-TCP within the initial time span after implantation, while later the tissue reaction was comparable to the HA-group [5,9] These and other different preclinical and clinical results substantiate the applicability of BBSM, as their biodegradation rate has shown to correlate with the process of bone tissue regeneration [10,11]. BBSM are assumed to be a suitable component of a BSP
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