Enhancement of bone healing through the direct injection of osteogenic and maintenance media concurrent with hydroxyapatite in a rat model of radial bone defects

Document Type : Full paper (Original article)

Authors

1 Ph.D. Student in Veterinary Surgery, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

3 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

10.22099/ijvr.2025.51865.7721

Abstract

Background: Healing critical bone defects remains a major challenge for orthopedic surgeons. Numerous studies have explored ways to enhance bone regeneration. Aims: This study aimed to evaluate the healing potential of osteogenic and maintenance cell culture media when applied directly to the defect site, either alone or in combination with hydroxyapatite (HA) granules. Methods: Seventy male Wistar rats were divided into seven groups: empty defect (n=10, negative control), autograft (n=10, positive control), HA (n=10), osteogenic medium (OM) (n=10), maintenance medium (MM) (n=10), MM+HA (n=10), and OM+HA (n=10). A 5 mm radial diaphyseal gap was created as a critical-sized defect. Culture media were injected transcutaneously into the gap on the fourth day post-surgery. Radiological evaluations were conducted on postoperative days 14, 28, 42, and 56. Histopathological assessments were performed on days 28 and 56. Results: OM and MM, both alone and combined with HA, demonstrated significant bone healing capacity. Radiologically, all treatment groups showed superior healing compared with the negative control. Bone regeneration in the OM group was comparable to the autograft group on days 42 and 56. Histopathologically, all groups exhibited advanced healing on days 28 and 56, except the HA group. Notably, bone healing in HA+OM and HA+MM on day 28, and in OM and MM on both days 28 and 56, was similar to that observed with autografts. Conclusion: Osteogenic and maintenance media exhibit promising bone regenerative properties and may serve as effective treatments for bone defects, provided that suitable delivery and support systems are developed.

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Main Subjects


Alesutan, I; Moritz, F; Haider, T; Shouxuan, S; Gollmann-Tepekoylu, C; Holfeld, J; Pieske, B; Lang, F; Eckardt, KU; Heinzmann, SS and Voelkl, J (2020). Impact of beta-glycerophosphate on the bioenergetic profile of vascular smooth muscle cells. J. Mol. Med. (Berl), 98: 985-997.
Alvarez-Elizondo, MB; Barenholz-Cohen, T and Weihs, D (2019). Sodium pyruvate pre-treatment prevents cell death due to localised, damaging mechanical strains in the context of pressure ulcers. Int. Wound J., 16: 1153-1163.
Asti, A; Gastaldi, G; Dorati, R; Saino, E; Conti, B; Visai, L and Benazzo, F (2010). Stem cells grown in osteogenic medium on PLGA, PLGA/HA, and titanium scaffolds for surgical applications. Bioinorg. Chem. Appl., 2010: 831031.
Attia, AK; Mahmoud, K; ElSweify, K; Bariteau, J and Labib, SA (2022). Donor site morbidity of calcaneal, distal tibial, and proximal tibial cancellous bone autografts in foot and ankle surgery. A systematic review and meta-analysis of 2296 bone grafts. Foot. Ankle. Surg., 28: 680-690.
Bellido, T; Plotkin, LI and Bruzzaniti, A (2014). Bone cells. In: Burr, DB and Allen, MR (Eds.), Basic and applied bone biology. (2nd Edn.), San Diego, United States, Academic Press. PP: 27-45.
Bigham-Sadegh, A; Mirshokraei, P; Karimi, I; Oryan, A; Aparviz, A and Shafiei-Sarvestani, Z (2012). Effects of adipose tissue stem cell concurrent with greater omentum on experimental long-bone healing in dog. Connect. Tissue Res., 53: 334-342.
Bigham-Sadegh, A; Torkestani, HS; Sharifi, S and Shirian, S (2020). Effects of concurrent use of royal jelly with hydroxyapatite on bone healing in rabbit model: radiological and histopathological evaluation. Heliyon. 6: e04547.
Blackwood, D and Seah, K (2009). Electrochemical cathodic deposition of hydroxyapatite: improvements in adhesion and crystallinity. Mater. Sci. Eng. C, 29: 1233-1238.
Bolander, ME and Balian, G (1986). The use of demineralized bone matrix in the repair of segmental defects. Augmentation with extracted matrix proteins and a comparison with autologous grafts. J. Bone. Joint. Surg. Am., 68: 1264-1274.
Borkowski, L; Jojczuk, M; Belcarz, A; Pawlowska-Olszewska, M; Kruk-Bachonko, J; Radzki, R; Bienko, M; Slowik, T; Lubek, T; Nogalski, A and Ginalska, G (2023). Comparing the healing abilities of fluorapatite and hydroxyapatite ceramics in regenerating bone tissue: An in vivo study. Materials (Basel). 16: 5992.
Buttery, L; Bielby, R; Howard, D and Shakesheff, K (2011). Osteogenic differentiation of embryonic stem cells in 2D and 3D culture. Methods Mol. Biol., 695: 281-308.
Camal Ruggieri, IN; Cicero, AM; Issa, JPM and Feldman, S (2021). Bone fracture healing: perspectives according to molecular basis. J. Bone. Miner. Metab., 39: 311-331.
Carpenter, RS; Goodrich, LR; Frisbie, DD; Kisiday, JD; Carbone, B; McIlwraith, CW; Centeno, CJ and Hidaka, C (2010). Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone. J. Orthop. Res., 28: 1330-1337.
Ciuffreda, MC; Malpasso, G; Musaro, P; Turco, V and Gnecchi, M (2016). Protocols for in vitro differentiation of human mesenchymal stem cells into osteogenic, chondrogenic and adipogenic lineages. Methods Mol. Biol., 1416: 149-158.
Cottrell, JA; Turner, JC; Arinzeh, TL and O'Connor, JP (2016). The biology of bone and ligament healing. Foot. Ankle Clin., 21: 739-761.
De Luca, A; Vitrano, I; Costa, V; Raimondi, L; Carina, V; Bellavia, D; Conoscenti, G; Di Falco, R; Pavia, FC; La Carrubba, V; Brucato, V and Giavaresi, G (2020). Improvement of osteogenic differentiation of human mesenchymal stem cells on composite poly l-lactic acid/nano-hydroxyapatite scaffolds for bone defect repair. J. Biosci. Bioeng., 129: 250-257.
Di Bella, C; Farlie, P and Penington, AJ (2008). Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng. Part A, 14: 483-490.
Dudas, JR; Marra, KG; Cooper, GM; Penascino, VM; Mooney, MP; Jiang, S; Rubin, JP and Losee, JE (2006). The osteogenic potential of adipose-derived stem cells for the repair of rabbit calvarial defects. Ann. Plast. Surg., 56: 543-548.
Emery, SE; Brazinski, MS; Koka, A; Bensusan, JS and Stevenson, S (1994). The biological and biomechanical effects of irradiation on anterior spinal bone grafts in a canine model. J. Bone. Joint Surg. Am., 76: 540-548.
Fan, L; Song, C; Lu, X; Wang, T; Han, J and Guo, R (2022). In situ preparation of hydroxyapatite in lamellar liquid crystals for joint lubrication and drug delivery. Soft. Matter. 18: 7859-7865.
Gasson, SB; Dobson, LK; Chow, L; Dow, S; Gregory, CA and Saunders, WB (2021). Optimizing in vitro osteogenesis in canine autologous and induced pluripotent stem cell-derived mesenchymal stromal cells with dexamethasone and BMP-2. Stem Cells. Dev., 30: 214-226.
Ghali, O; Broux, O; Falgayrac, G; Haren, N; van Leeuwen, JP; Penel, G; Hardouin, P and Chauveau, C (2015). Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation. BMC Cell Biol., 16: 9.
Hata, R and Senoo, H (1989). L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts. J. Cell. Physiol., 138: 8-16.
Hinoi, E; Fujimori, S; Takemori, A and Yoneda, Y (2002). Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts. Biochem. Biophys. Res. Commun., 294: 1177-1183.
Huang, YM; Lin, YC; Chen, CY; Hsieh, YY; Liaw, CK; Huang, SW; Tsuang, YH; Chen, CH and Lin, FH (2020). Thermosensitive chitosan-gelatin-glycerol phosphate hydrogels as collagenase carrier for tendon-bone healing in a rabbit model. Polymers (Basel). 12: 436.
Jamal, MS; Hurley, ET; Asad, H; Asad, A and Taneja, T (2022). The role of platelet rich plasma and other orthobiologics in bone healing and fracture management: A systematic review. J. Clin. Orthop. Trauma. 25: 101759.
Ji, X; Yuan, X; Ma, L; Bi, B; Zhu, H; Lei, Z; Liu, W; Pu, H; Jiang, J; Jiang, X; Zhang, Y and Xiao, J (2020). Mesenchymal stem cell-loaded thermosensitive hydroxypropyl chitin hydrogel combined with a three-dimensional-printed poly (epsilon-caprolactone)/nano-hydroxyapatite scaffold to repair bone defects via osteogenesis, angiogenesis and immunomodulation. Theranostics. 10: 725-740.
Khotib, J; Gani, MA; Budiatin, AS; Lestari, M; Rahadiansyah, E and Ardianto, C (2021). Signaling pathway and transcriptional regulation in osteoblasts during bone healing: Direct involvement of hydroxyapatite as a biomaterial. Pharmaceuticals (Basel). 14: 615.
Kim, JH and Kim, HW (2013). Rat defect models for bone grafts and tissue engineered bone constructs. J. Tissue Eng. Regen. Med., 10: 310-316.
Lane, JM and Sandhu, HS (1987). Current approaches to experimental bone grafting. Orthop. Clin. North Am., 18: 213-225.
Langenbach, F and Handschel, J (2013). Effects of dexamethasone, ascorbic acid and beta-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res. Ther., 4: 117.
Lee, JB; Kim, JE; Balikov, DA; Bae, MS; Heo, DN; Lee, D; Rim, HJ; Lee, DW; Sung, HJ and Kwon, IK (2016). Poly (l-lactic acid)/Gelatin fibrous scaffold loaded with simvastatin/Beta-cyclodextrin-modified hydroxyapatite inclusion complex for bone tissue regeneration. Macromol. Biosci., 16: 1027-1038.
Li, R; Li, D; Wang, H; Chen, K; Wang, S; Xu, J and Ji, P (2022). Exosomes from adipose-derived stem cells regulate M1/M2 macrophage phenotypic polarization to promote bone healing via miR-451a/MIF. Stem Cell Res. Ther., 13: 149.
Lin, CY; Chang, YH; Li, KC; Lu, CH; Sung, LY; Yeh, CL; Lin, KJ; Huang, SF; Yen, TC and Hu, YC (2013). The use of ASCs engineered to express BMP2 or TGF-beta3 within scaffold constructs to promote calvarial bone repair. Biomaterials, 34: 9401-9412.
Lin, L; Chow, KL and Leng, Y (2009). Study of hydroxyapatite osteoinductivity with an osteogenic differentiation of mesenchymal stem cells. J. Biomed. Mater. Res. Part A. 89: 326-335.
Liu, Y; Puthia, M; Sheehy, EJ; Ambite, I; Petrlova, J; Prithviraj, S; Oxborg, MW; Sebastian, S; Vater, C; Zwingenberger, S; Struglics, A; Bourgine, PE; O'Brien, FJ and Raina, DB (2023). Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing. Acta Biomater., 162: 164-181.
Lv, Q; Nair, L and Laurencin, CT (2009). Fabrication, characterization, and in vitro evaluation of poly (lactic acid glycolic acid)/nano-hydroxyapatite composite microsphere-based scaffolds for bone tissue engineering in rotating bioreactors. J. Biomed. Mater. Res. Part A. 91: 679-691.
Mai, TP; Park, JB; Nguyen, HD; Min, KA and Moon, C (2023). Current application of dexamethasone-incorporated drug delivery systems for enhancing bone formation. J. Pharm. Investig., 53: 643-665.
Marsell, R and Einhorn, TA (2011). The biology of fracture healing. Injury. 42: 551-555.
Martin, MJ; Muotri, A; Gage, F and Varki, A (2005). Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med., 11: 228-232.
Mayfield, CK; Ayad, M; Lechtholz-Zey, E; Chen, Y and Lieberman, JR (2022). 3D-Printing for critical sized bone defects: Current concepts and future directions. Bioengineering (Basel). 9: 680.
Miller, CP and Chiodo, CP (2016). Autologous bone graft in foot and ankle surgery. Foot. Ankle Clin., 21: 825-837.
Mori, K; Shioi, A; Jono, S; Nishizawa, Y and Morii, H (1999). Dexamethasone enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol., 19: 2112-2118.
Moriguchi, N; Hinoi, E; Tsuchihashi, Y; Fujimori, S; Iemata, M; Takarada, T and Yoneda, Y (2006). Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts. Histol. Histopathol., 7/9: 0213-3911
Morsczeck, C and Reichert, TE (2017). The dexamethasone induced osteogenic differentiation of dental follicle cells. Histol. Histopathol., 32: 1223-1229.
Munir, MU; Salman, S; Javed, I; Bukhari, SNA; Ahmad, N; Shad, NA and Aziz, F (2021). Nano-hydroxyapatite as a delivery system: overview and advancements. Artif. Cells Nanomed. Biotechnol., 49: 717-727.
O'Hare, P; Meenan, BJ; Burke, GA; Byrne, G; Dowling, D and Hunt, JA (2010). Biological responses to hydroxyapatite surfaces deposited via a co-incident microblasting technique. Biomaterials, 31: 515-522.
Oliveira, HL; Da Rosa, WLO; Cuevas-Suarez, CE; Carreno, NLV; da Silva, AF; Guim, TN; Dellagostin, OA and Piva, E (2017). Histological evaluation of bone repair with hydroxyapatite: A systematic review. Calcif. Tissue Int., 101: 341-354.
Oryan, A; Alidadi, S and Moshiri, A (2016). Platelet-rich plasma for bone healing and regeneration. Expert Opin. Biol. Ther., 16: 213-232.
Oryan, A; Bigham-Sadegh, A and Abbasi-Teshnizi, F (2014). Effects of osteogenic medium on healing of the experimental critical bone defect in a rabbit model. Bone, 63: 53-60.
Oryan, A; Meimandi Parizi, A; Shafiei-Sarvestani, Z and Bigham, AS (2012). Effects of combined hydroxyapatite and human platelet rich plasma on bone healing in rabbit model: radiological, macroscopical, hidtopathological and biomechanical evaluation. Cell. Tissue Bank., 13: 639-651.
Oryan, A; Monazzah, S and Bigham-Sadegh, A (2015). Bone injury and fracture healing biology. Biomed. Environ. Sci., 28: 57-71.
Persson, M; Lehenkari, PP; Berglin, L; Turunen, S; Finnila, MAJ; Risteli, J; Skrifvars, M and Tuukkanen, J (2018). Osteogenic differentiation of human mesenchymal stem cells in a 3D woven scaffold. Sci. Rep., 8: 10457.
Polat, O; Kilicoglu, SS and Erdemli, E (2007). A controlled trial of glutamine effects on bone healing. Adv. Ther., 24: 154-160.
Rajewska, J; Kowalski, J; Matys, J; Dobrzynski, M and Wiglusz, RJ (2023). The use of lactide polymers in bone tissue regeneration in dentistry-A systematic review. J. Funct. Biomater., 14: 83.
Roddy, E; DeBaun, MR; Daoud-Gray, A; Yang, YP and Gardner, MJ (2018). Treatment of critical-sized bone defects: clinical and tissue engineering perspectives. Eur. J. Orthop. Surg. Traumatol., 28: 351-362.
Roseti, L; Parisi, V; Petretta, M; Cavallo, C; Desando, G; Bartolotti, I and Grigolo, B (2017). Scaffolds for bone tissue engineering: State of the art and new perspectives. Mater. Sci. Eng. C Mater. Biol. Appl., 78: 1246-1262.
Sadat-Shojai, M; Khorasani, MT; Dinpanah-Khoshdargi, E and Jamshidi, A (2013). Synthesis methods for nanosized hydroxyapatite with diverse structures. Acta Biomater., 9: 7591-7621.
Sandor, GK; Numminen, J; Wolff, J; Thesleff, T; Miettinen, A; Tuovinen, VJ; Mannerstrom, B; Patrikoski, M; Seppanen, R; Miettinen, S; Rautiainen, M and Ohman, J (2014). Adipose stem cells used to reconstruct 13 cases with cranio-maxillofacial hard-tissue defects. Stem Cells Transl. Med., 3: 530-540.
Sattary, M; Rafienia, M; Kazemi, M; Salehi, H and Mahmoudzadeh, M (2019). Promoting effect of nano hydroxyapatite and vitamin D3 on the osteogenic differentiation of human adipose-derived stem cells in polycaprolactone/gelatin scaffold for bone tissue engineering. Mater. Sci. Eng. C Mater. Biol. Appl., 97: 141-155.
Schack, LM; Noack, S; Winkler, R; Wissmann, G; Behrens, P; Wellmann, M; Jagodzinski, M; Krettek, C and Hoffmann, A (2013). The phosphate source influences gene expression and quality of mineralization during in vitro osteogenic differentiation of human mesenchymal stem cells. PLoS One. 8: e65943.
Seol, YJ; Kim, JY; Park, EK; Kim, SY and Cho, DW (2009). Fabrication of a hydroxyapatite scaffold for bone tissue regeneration using microstereolithography and molding technology. Microelectron. Eng., 86: 1443-1446.
Shafiei, Z; Bigham, AS; Dehghani, SN and Nezhad, ST (2009). Fresh cortical autograft versus fresh cortical allograft effects on experimental bone healing in rabbits: radiological, histopathological and biomechanical evaluation. Cell. Tissue Bank., 10: 19-26.
Shafiei-Sarvestani, Z; Oryan, A; Bigham, AS and Meimandi-Parizi, A (2012). The effect of hydroxyapatite-hPRP, and coral-hPRP on bone healing in rabbits: radiological, biomechanical, macroscopic and histopathologic evaluation. Int. J. Surg., 10: 96-101.
Shibli, JA; Nagay, BE; Suarez, LJ; Urdaniga Hung, C; Bertolini, M; Barao, VAR and Souza, JGS (2022). Bone tissue engineering using osteogenic cells: From the bench to the clinical application. Tissue Eng. Part C Methods. 28: 179-192.
Shioi, A; Nishizawa, Y; Jono, S; Koyama, H; Hosoi, M and Morii, H (1995). Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol., 15: 2003-2009.
Sundin, M; Ringden, O; Sundberg, B; Nava, S;
Gotherstrom, C and Le Blanc, K
(2007). No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica, 92: 1208-1215.
Takamizawa, S; Maehata, Y; Imai, K; Senoo, H; Sato, S and Hata, R (2004). Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol. Int., 28: 255-265.
Tirkkonen, L; Haimi, S; Huttunen, S; Wolff, J; Pirhonen, E; Sándor, G and Miettinen, S (2013). Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture. Eur. Cell. Mater., 25: e58.
Trombelli, L; Simonelli, A; Pramstraller, M; Wikesjö, UM and Farina, R (2010). Single flap approach with and without guided tissue regeneration and a hydroxyapatite biomaterial in the management of intraosseous periodontal defects. J. Periodontol., 81: 1256-1263.
Vater, C; Kasten, P and Stiehler, M (2011). Culture media for the differentiation of mesenchymal stromal cells. Acta Biomater., 7: 463-477.
Verrier, S; Alini, M; Alsberg, E; Buchman, SR; Kelly, D; Laschke, MW; Menger, MD; Murphy, WL; Stegemann, JP; Schutz, M; Miclau, T; Stoddart, MJ and Evans, C (2016). Tissue engineering and regenerative approaches to improving the healing of large bone defects. Eur. Cell. Mater., 32: 87-110.
Yang, X; Li, Y; Liu, X; Zhang, R and Feng, Q (2018). In vitro uptake of hydroxyapatite nanoparticles and their effect on osteogenic differentiation of human mesenchymal stem cells. Stem Cells Int., 2018: 2036176.
Ye, Q; Ohsaki, K; Li, K; Li, DJ; Zhu, CS; Ogawa, T; Tenshin, S and Takano-Yamamoto, T (2001). Histological reaction to hydroxyapatite in the middle ear of rats. Auris Nasus Larynx. 28: 131-136.
Yevlashevskaya, OS; Scheven, BA; Walmsley, AD and Shelton, RM (2023). Differing responses of osteogenic cell lines to beta-glycerophosphate. Sci. Rep., 13: 14472.
Yi, Y; Wu, M; Zhou, X; Xiong, M; Tan, Y; Yu, H; Liu, Z; Wu, Y and Zhang, Q (2022). Ascorbic acid 2-glucoside preconditioning enhances the ability of bone marrow mesenchymal stem cells in promoting wound healing. Stem Cell Res. Ther., 13: 119.
Yoon, E; Dhar, S; Chun, DE; Gharibjanian, NA and Evans, GR (2007). In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng., 13: 619-627.
Yuasa, M; Yamada, T; Taniyama, T; Masaoka, T; Xuetao, W; Yoshii, T; Horie, M; Yasuda, H; Uemura, T; Okawa, A and Sotome, S (2015). Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2. PLoS One. 10: e0116462.