Histomorphometric analysis of biological responses following the use of pure hydroxyapatite and hydroxyapatite with collagen: a study in dorsal subcutaneous tissue of rats / Análise histomorfométrica das respostas biológicas após o uso de hidroxiapatita pura e hidroxiapatita com colágeno: um estudo no tecido subcutâneo dorsal de ratos

Dr Fabio Anibal Goiris, Dr Michel Fleith Otuki, Dr Sandra Regina Antunes, Dr Fábio André dos Santos, Dr Juliana Larocca de Geus, Dr Eduardo Bauml Campagnoli, Dr Edson Durval Menezes Alves, DDS Érika de Lara, DDS Matheus Tadao Wakasugui, DDS Roberto Nakakogue


Purpose: The aim of this study was to evaluate the biological responses resulting from the implantation of two types of experimental hydroxyapatite – Pure Ha (HaP) and Ha with collagen (HaCol) and compare them with a third type HaAlobone (commercial) – on dorsal subcutaneous tissue of female rats. Methods: Forty-five animals were used (15 in each group), which were sacrificed 7, 15, and 30 days after operation. The specimens were fixed, stained with hematoxylin and eosin, and then evaluated for inflammatory reactions with a light microscope. Results: The three experimental groups showed a high inflammatory response after 7 days. The inflammatory response was seen to decrease sharply after 15 days. After 30 days, the foreign body reactions were seen to reduce significantly, and an organized collagen tissue was observed. The results showed that the types of hydroxyapatite tested – HaP and HaCol – are biocompatible. When compared with the commercially available hydroxyapatite, these new biomaterials showed similar biocompatibility performance.

CLINICAL SIGNIFICANCE: The new hydroxyapatite tested are considered biocompatible.


Hydroxyapatites, Materials Testing, In Vitro Techniques.

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Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury 2005; 36 : S20-S27.

Sun D, Chen Y, Tran RT, Xu S, Xie D, Jia C, Wang Y, Guo Y, Yang J, Jin D, Bai X. Citric acid-based hydroxyapatite composite scaffolds enhance calvarial regeneration. Sci Rep 2014; 4: 6912.

Breeze J, Patel J, Dover MS, Williams RW. Success rates and complications of autologous onlay bone grafts and sinus lifts in patients with congenital hypodontia and after trauma. Br J Oral Maxillofac Surg 2017; 55: 830-833.

Oyane A. Development of apatite-based composites by a biomimetic process for biomedical appications. J Ceram Soc Jpn 2010; 118: 77-81.

Yoshikawa H, Tamai N, Murase T, Myoui A. Interconnected porous hydroxyapatite ceramics for bone tissue engineering. J R Soc Interface 2009; 6: S341-S348.

Ramesh N, Moratti SC, Dias GJ. Hydroxyapatite-polymer biocomposites for bone regeneration: A review of current trends. J Biomed Mater Res B Appl Biomater 2018; 106: 2046-2057.

Yoshikawa H, Myoui A. Bone tissue engineering with porous hydroxyapatite ceramics. J Artif Organs 2005; 8: 131-136.

Artzi Z, Weinreb M, Givol N, Rohrer MD, Nemcovsky CE, Prasad HS, Tal H. Biomaterial resorption rate and healing site morphology of inorganic bovine bone and beta-tricalcium phosphate in the canine: a 24-month longitudinal histologic study and morphometric analysis. Int J Oral Maxillofac Implants 2004; 19: 357-368.

Sun F, Zhou H, Lee J. Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites for bone regeneration. Acta Biomater 2011; 7: 3813-3828.

An YH, Woolf SK, Friedman RJ. Pre-clinical in vivo evaluation of orthopaedic bioabsorbable devices. Biomaterials 2000; 21: 2635-2652.

Kim J, Dadsetan M, Ameenuddin S, Windebank AJ, Yaszemski MJ, Lu L. In vivo biodegradation and biocompatibility of PEG/sebacic acid-based hydrogels using a cage implant system. J Biomed Mater Res A 2010; 95: 191-197.

Yin Y, Ye F, Cui J, Zhang F, Li X, Yao K. Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphate composite scaffolds for bone tissue engineering. J Biomed Mater Res A 2003; 67: 844-855.

El-Rouby DH, Halim SAE. A comparative histological, histochemical, and immunohistochemical study of the biocompatibility of three different nano-restorative material implanted in rats’ connective tissue. Conference Proceedings 2005 : 1-23.

da Cruz AC, Pochapski MT, Daher JB, da Silva JC, Pilatti GL, Santos FA. Physico-chemical characterization and biocompatibility evaluation of hydroxyapatites. J Oral Sci 2006; 48: 219-226.

Tomida M, Nakano K, Matsuura S, Kawakami T. Comparative examination of subcutaneous tissue reaction to high molecular materials in medical use. Eur J Med Res 2011; 16: 249-252.

Massari KV, Marinho GO, Silva JL, Holgado LA, Leão AL, Chaves MRM, Kinoshita A. Tissue reaction after subcutaneous implantation of a membrane composed of bacterial cellulose embedded with hydroxyapatite. Dent Oral Craniofac Res 2015;1 : 25-30.

Xie L, Yu H, Yang W, Zhu Z, Yue L. Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds. J Biomater Sci Polym Ed 2016; 27: 505-528.

Silva CC, Pinheiro AG, Miranda MAR, Góes JC, Sombra ASB. Structural properties of hydroxyapatite obtained by mechanosynthesis. Solid State Sci 2003; 5: 553-558.

Mezadri TJ TV, Amaral VLL. Anestesia e analgesia em animais de laboratório. In: Animais de laboratório: cuidados na iniciação experimental. Editora da UFSC; Florianopólis 2004; 101-130.

Hefti AF, Preshaw PM. Examiner alignment and assessment in clinical periodontal research. Periodontol 2000 2012; 59: 41-60.

Zaazou MH, Zaki DY, Mostafa AA, Mahmoud AA, Basha M, Khallaf M. In vivo biocompatibility evaluation of injectable nano-hydroxyapatite/ Poloxamer 407 based formulations. Part 1. J app Sci Res 2013; 9: 5269-5276.

Conz MB, Granjeiro JM, Soares Gde A. Physicochemical characterization of six commercial hydroxyapatites for medical-dental applicatons as bone graft. J Appl Oral Sci 2005; 13: 136-140.

Federation Dentaire International, Commission of Dental Materials, Instruments, Equipment and Therapeutics. Recommended standard practices for biological evaluation of dental materials. Int Dent J 1980; 30: 140-188.

Afnan MAM, Saxena AK. Tissue repair in neonatal and paediatric surgery: Analysis of infection in surgical implantation of synthetic resorbable biomaterials. Biomed Mater Eng 2018; 29: 799-808.

Benetti F, de Azevedo Queiroz IO, Oliveira PHC, Conti LC, Azuma MM, Oliveira SHP, Cintra LTA. Cytotoxicity and biocompatibility of a new bioceramic endodontic sealer containing calcium hydroxide. Braz Oral Res 2019; 33: e042.

Huang X, Miao X. Novel porous hydroxyapatite prepared by combining H2O2 foaming with PU sponge and modified with PLGA and bioactive glass. J Biomater Appl 2007; 21: 351-374

Seyednejad H, Gawlitta D, Kuiper RV, de Bruin A, van Nostrum CF, Vermonden T, Dhert WJ, Hennink WE. In vivo biocompatibility and biodegradation of 3D-printed porous scaffolds based on a hydroxyl-functionalized poly(epsilon-caprolactone). Biomaterials 2012; 33: 4309-4318.

Wahl DA, Czernuszka JT. Collagen-hydroxyapatite composites for hard tissue repair. Eur Cell Mater 2006; 11: 43-56.

Isikli C, Hasirci V, Hasirci N. Development of porous chitosan-gelatin/hydroxyapatite composite scaffolds for hard tissue-engineering applications. J Tissue Eng Regen Med 2012; 6: 135-143.

Arafat MT, Lam CX, Ekaputra AK, Wong SY, Li X, Gibson I. Biomimetic composite coating on rapid prototyped scaffolds for bone tissue engineering. Acta Biomater 2011; 7: 809-820.

Wang K, Zhou C, Hong Y, Zhang X. A review of protein adsorption on bioceramics. Interface Focus 2012; 2: 259-277.

Sawyer AA, Hennessy KM, Bellis SL. Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins. Biomaterials 2005; 26: 1467-1475.

Vishwakarma A, Bhise NS, Evangelista MB, Rouwkema J, Dokmeci MR, Ghaemmaghami AM, et al. Engineering Immunomodulatory Biomaterials To Tune the Inflammatory Response. Trends Biotechnol 2016; 34: 470-482.

Ge Z, Baguenard S, Lim LY, Wee A, Khor E. Hydroxyapatite-chitin materials as potential tissue engineered bone substitutes. Biomaterials 2004; 25: 1049-1058.

Legrand AP, Marinov G, Pavlov S, Guidoin MF, Famery R, Bresson B, Zhang Z, Guidoin R. Degenerative mineralization in the fibrous capsule of silicone breast implants. J Mater Sci Mater Med 2005; 16: 477-485.

Khashaba RM, Moussa MM, Mettenburg DJ, Rueggeberg FA, Chutkan NB, Borke JL. Polymeric-calcium phosphate cement composites-material properties: in vitro and in vivo investigations. Int J Biomater 2010; 2010: 691452.

van der Meulen J, Koerten HK. Inflammatory response and degradation of three types of calcium phosphate ceramic in a non-osseous environment. J Biomed Mater Res 1994; 28: 1455-1463.

Fang CH, Lin YW, Lin FH, Sun JS, Chao YH, Lin HY, Chang ZC. Biomimetic Synthesis of Nanocrystalline Hydroxyapatite Composites: Therapeutic Potential and Effects on Bone Regeneration. Int J Mol Sci 2019; 20: 6002.

Bracken MB. Why animal studies are often poor predictors of human reactions to exposure. J R Soc Med 2009; 102: 120-122.

Jayasree R, Kumar TSS, Venkateswari R, Nankar RP, Doble M. Eggshell derived brushite bone cement with minimal inflammatory response and higher osteoconductive potential. J Mater Sci Mater Med 2019; 30: 113.

DOI: https://doi.org/10.34117/bjdv7n2-082


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