Bio oss small

The five experimental groups of eight animals were subjected to critical bone defects of 8 mm, performed with trephine in the rats’ calvaria and the groups according to the treatment method they were assigned as:
Control group—there was no biomaterial placement in the critical bone defects, only natural clot.
Group 1—the critical bone defects were filled with synthetic hydroxyapatite, 0.10 g of (Alobone poros Osseocon Biomateriais Ltd.a., Rio de Janeiro/RJ, Brazil).
Group 2—the critical bone defects were filled with xenograft, 0.10 g of (Bio-Oss^® Small Geistlich Pharma AG, Wolhusen, Switzerland).
Group 3—the critical bone defects were filled with the same synthetic hydroxyapatite used in group 1, with the same weight of 0.10 g (Alobone poros™, Osseocon Biomateriais Ltd.a., Rio de Janeiro/RJ, Brazil), enriched with 300 μL of BMMF 1 × 10⁶, obtained from marrow donor animals. (Figure 1a–c).
Group 4—the critical bone defects were filled with the same xenograft, used in group 2, with the same weight of 0.10 g (Bio-Oss Small™, Geistlich Pharma AG, Wolhusen, Switzerland), enriched with 300 μL of BMMF 1 × 10⁶ obtained from the other four marrow donor animals.

Twenty-four male Wistar rats (Rattus norvegicus albinus) were used in this study, with prior approval from the Research Ethics Committee for Animal Experimentation of the Faculdade São Leopoldo Mandic (ethical protocol approval n. 2020/010). The study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiment) guidelines. Considering the possibility of human cell rejection, an immunosuppression protocol was started 10 days before the experiment, as described by Lekhooa et al. (2017) [27 (link)], and followed until euthanasia. The animals were kept under controlled conditions of temperature and lighting, with a 12-h light–dark cycle, balanced food, and water ad libitum.
The animals were randomly divided into two groups, according to the treatment: Group 1 (G1) and Group 2 (G2). In G1 (n = 12), bone defects were filled with Bio-Oss xenograft (Bio-Oss^® small—Geistlich Pharma AG, Wolhusen, Switzerland) and covered by collagen membrane (Bio-Gide^®—Geistlich Pharma AG, Wolhusen, Switzerland). In G2 (n = 12), bone defects were filled by Bio-Oss^® small particles in addition to conditioned MSCs culture medium (CM-MSCs) prepared as described below and covered by Bio-Gide^® collagen membrane. The animals were euthanized after 14 days (T1) or 42 days (T2).

The freeze-dried conditioned medium in powder form was stored at −80 °C for up to 3 months. At the time of experiment, the lyophilized powder was dissolved in deionized water at a concentration of 20 mg/mL and then filtered through a 0.2 μm filter to produce a sterile final product, and then used right away.
The scaffold used in this study was the Bio-Oss bone substitute bimaterial (Bio-Oss^® small—Geistlich Pharma AG, Wolhusen, Switzerland). It is a xenograft hydroxyapatite of bovine origin that is commonly used clinically to fill bone defects. Sequentially, 100 mg of xenograft granules was drip-associated with the reconstituted lyophilized culture medium in 12-well cell culture plates (Corning, New York, NY, USA) immediately before the surgery procedure.