Biopolymer composites allow the creation of an optimal environment for the regeneration of chondral and osteochondral defects of articular cartilage, where natural regeneration potential is limited. In this experimental study, we used the sheep animal model for the creation of knee cartilage defects. In the medial part of the trochlea and on the medial condyle of the femur, we created artificial defects (6 × 3 mm2) with microfractures. In four experimental sheep, both defects were subsequently filled with the porous acellular polyhydroxybutyrate/chitosan (PHB/CHIT)-based implant. Two sheep had untreated defects. We evaluated the quality of the newly formed tissue in the femoral trochlea defect site using imaging (X-ray, Computer Tomography (CT), Magnetic Resonance Imaging (MRI)), macroscopic, and histological methods. Macroscopically, the surface of the treated regenerate corresponded to the niveau of the surrounding cartilage. X-ray examination 6 months after the implantation confirmed the restoration of the contour in the subchondral calcified layer and the advanced rate of bone tissue integration. The CT scan revealed a low regenerative potential in the bone zone of the defect compared to the cartilage zone. The percentage change in cartilage density at the defect site was not significantly different to the reference area (0.06-6.4%). MRI examination revealed that the healing osteochondral defect was comparable to the intact cartilage signal on the surface of the defect. Hyaline-like cartilage was observed in most of the treated animals, except for one, where the defect was repaired with fibrocartilage. Thus, the acellular, chitosan-based biomaterial is a promising biopolymer composite for the treatment of chondral and osteochondral defects of traumatic character. It has potential for further clinical testing in the orthopedic field, primarily with the combination of supporting factors.

Center of Clinical and Preclinical Research MediPark Pavol Jozef Safarik University 04011 Kosice Slovakia

Clinic of Horses University of Veterinary Medicine and Pharmacy in Kosice Komenskeho 73 04181 Kosice Slovakia

Department of Biology and Physiology University of Veterinary Medicine and Pharmacy in Kosice Komenskeho 73 04181 Kosice Slovakia

Department of Biomedical Engineering and Measurement Faculty of Mechanical Engineering Technical University of Kosice Letna 9 04200 Kosice Slovakia

Department of Biomedical Research East Slovak Institute of Cardiovascular Diseases Inc 04011 Kosice Slovakia

Department of Morphological Disciplines University of Veterinary Medicine and Pharmacy in Kosice Komenskeho 73 04181 Kosice Slovakia

Institute of Anatomy Charles University U nemocnice 3 12800 Prague Czech Republic

Institute of Materials Research The Slovak Academy of Sciences Watsonova 1935 47 04001 Kosice Slovakia

Institute of Physiology The Czech Academy of Sciences Videnska 1083 14200 Prague Czech Republic

Prague Burn Centre 3rd Faculty of Medicine Charles University and University Hospital Kralovske Vinohrady 10034 Prague Czech Republic

Railway Hospital in Kosice Masarykova 1632 9 04001 Kosice Slovakia

See more in PubMed

Ross M.H., Pawlina W. Histology: A Text and Atlas. 6th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins; Baltimore, MA, USA: 2011. pp. 198–207.

Karuppal R. Current concepts in the articular cartilage repair and regeneration. J. Orthop. 2017;14:A1–A3. doi: 10.1016/j.jor.2017.05.001. PubMed DOI PMC

Musumeci G. New perspectives for articular cartilage repair treatment through tissue engineering: A contemporary review. World J. Orthop. 2014;5:80–88. doi: 10.5312/wjo.v5.i2.80. PubMed DOI PMC

Poulet B. Models to define the stages of articular cartilage degradation in osteoarthritis development. Int. J. Exp. Pathol. 2017;98:120–126. doi: 10.1111/iep.12230. PubMed DOI PMC

Music E., Futrega K., Doran M. Sheep as a model for evaluating mesenchymal stem/stromal cell (MSC)-based chondral defect repair. Osteoarthr. Cartil. 2018;26:730–740. doi: 10.1016/j.joca.2018.03.006. PubMed DOI

Neidlin M., Chantzi E., Macheras G., Gustafsson M.G., Alexopoulos L.G. An ex vivo tissue model of cartilage degradation suggests that cartilage state can be determined from secreted key protein patterns. PLoS ONE. 2019;14:e0224231. doi: 10.1371/journal.pone.0224231. PubMed DOI PMC

Berenbaum F., Wallace I.J., Lieberman D.E., Felson D.T. Modern-day environmental factors in the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 2018;14:674–681. doi: 10.1038/s41584-018-0073-x. PubMed DOI

Bortoluzzi A., Furini F., Scirè C.A. Osteoarthritis and its management—Epidemiology, nutritional aspects and environmental factors. Autoimmun. Rev. 2018;17:1097–1104. doi: 10.1016/j.autrev.2018.06.002. PubMed DOI

Medvedeva E.V., Grebenik E.A., Gornostaeva S.N., Telpuhov V.I., Lychagin A.V., Timashev P.S., Chagin A.S. Repair of Damaged Articular Cartilage: Current Approaches and Future Directions. Int. J. Mol. Sci. 2018;19:2366. doi: 10.3390/ijms19082366. PubMed DOI PMC

Redman S.N., Oldfield S.F., Archer C.W. Current strategies for articular cartilage repair. Eur. Cells Mater. 2005;9:23–32. doi: 10.22203/eCM.v009a04. PubMed DOI

Chu C.R., Szczodry M., Bruno S. Animal Models for Cartilage Regeneration and Repair. Tissue Eng. Part B Rev. 2010;16:105–115. doi: 10.1089/ten.teb.2009.0452. PubMed DOI PMC

Dias I.R., Viegas C.A., Carvalho P.P. Large Animal Models for Osteochondral Regeneration. Tissue Eng. 2018;1059:441–501. doi: 10.1007/978-3-319-76735-2_20. PubMed DOI

Hoemann C.D., Hurtig M., Rossomacha E., Sun J., Chevrier A., Shive M.S., Buschmann M.D. Chitosan-Glycerol Phosphate/Blood Implants Improve Hyaline Cartilage Repair in Ovine Microfracture Defects. J. Bone Jt. Surg. Am. Vol. 2005;87:2671–2686. doi: 10.2106/00004623-200512000-00011. PubMed DOI

Hao T., Wen N., Cao J.-K., Wang H.-B., Lü S.-H., Liu T., Lin Q.-X., Duan C.-M., Wang C.-Y. The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels. Osteoarthr. Cartil. 2010;18:257–265. doi: 10.1016/j.joca.2009.08.007. PubMed DOI

Zhu C., Wu Q., Wang F., Zhang X., Chen F., Liu X., Yang Q., Zhu L. Animal Models Used for Testing Hydrogels in Cartilage Regeneration. Curr. Stem Cell Res. Ther. 2018;13:517–525. doi: 10.2174/1574888X13666180514123103. PubMed DOI

Kim I.-Y., Seo S.-J., Moon H.-S., Yoo M.-K., Park I.-Y., Kim B.-C., Cho C.-S. Chitosan and its derivatives for tissue engineering applications. Biotechnol. Adv. 2008;26:1–21. doi: 10.1016/j.biotechadv.2007.07.009. PubMed DOI

Rogina A., Pušić M., Štefan L., Ivković A., Urlić I., Ivanković M., Ivanković H. Characterization of Chitosan-Based Scaffolds Seeded with Sheep Nasal Chondrocytes for Cartilage Tissue Engineering. Ann. Biomed. Eng. 2021:1–15. doi: 10.1007/s10439-020-02712-9. PubMed DOI

Victor R.D.S., Santos A.M.D.C., De Sousa B.V., Neves G.D.A., Santana L.N.D.L., Menezes R.R. A Review on Chitosan’s Uses as Biomaterial: Tissue Engineering, Drug Delivery Systems and Cancer Treatment. Materials. 2020;13:4995. doi: 10.3390/ma13214995. PubMed DOI PMC

Concha M., Vidal A., Giacaman A., Ojeda J., Pavicic F., Oyarzun-Ampuero F.A., Torres C., Cabrera M., Moreno-Villoslada I., Orellana S.L. Aerogels made of chitosan and chondroitin sulfate at high degree of neutralization: Biological properties toward wound healing. J. Biomed. Mater. Res. Part B Appl. Biomater. 2018;106:2464–2471. doi: 10.1002/jbm.b.34038. PubMed DOI

Oryan A., Sahvieh S. Effectiveness of chitosan scaffold in skin, bone and cartilage healing. Int. J. Biol. Macromol. 2017;104:1003–1011. doi: 10.1016/j.ijbiomac.2017.06.124. PubMed DOI

Oprenyeszk F., Sanchez C., Dubuc J.-É., Maquet V., Henrist C., Compère P., Henrotin Y. Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes. PLoS ONE. 2015;10:e0128362. doi: 10.1371/journal.pone.0128362. PubMed DOI PMC

Comblain F., Rocasalbas G., Gauthier S., Henrotin Y. Chitosan: A promising polymer for cartilage repair and viscosupplementation. Bio Med. Mater. Eng. 2017;28:S209–S215. doi: 10.3233/BME-171643. PubMed DOI

Islam M., Shahruzzaman M., Biswas S., Sakib N., Rashid T.U. Chitosan based bioactive materials in tissue engineering applications-A review. Bioact. Mater. 2020;5:164–183. doi: 10.1016/j.bioactmat.2020.01.012. PubMed DOI PMC

Hill R.G. Biomaterials, Artificial Organs and Tissue Engineering. Elsevier; Amsterdam, The Netherlands: 2005. Biomedical polymers; pp. 97–106.

Dariš B., Knez Ž. Poly(3-hydroxybutyrate): Promising biomaterial for bone tissue engineering. Acta Pharm. 2020;70:1–15. doi: 10.2478/acph-2020-0007. PubMed DOI

Giretova M., Medvecky L., Petrovova E., Cizkova D., Danko J., Mudronova D., Slovinska L., Bures R. Polyhydroxybutyrate/Chitosan 3D Scaffolds Promote In Vitro and In Vivo Chondrogenesis. Appl. Biochem. Biotechnol. 2019;189:556–575. doi: 10.1007/s12010-019-03021-1. PubMed DOI

Kok A.C., van Bergen C.J.A., Tuijthof G.J.M., Klinkenbijl M.N., van Noorden C.J.F., van Dijk C.N., Kerkhoffs G.M.M.J. Macroscopic ICRS Poorly Correlates with O’Driscoll Histological Cartilage Repair Assessment in a Goat Model. Clin. Res. Foot Ankle. 2015;3:1–7. doi: 10.4172/2329-910x.1000173. DOI

Moojen D., Saris D., Yang K.A., Dhert W., Verbout A. The Correlation and Reproducibility of Histological Scoring Systems in Cartilage Repair. Tissue Eng. 2002;8:627–634. doi: 10.1089/107632702760240544. PubMed DOI

Orth P., Madry H. Complex and elementary histological scoring systems for articular cartilage repair. Histol. Histopathol. 2015;30:911–919. PubMed

Medvecky L., Giretova M., Stulajterova R. Properties and in vitro characterization of polyhydroxybutyrate–chitosan scaffolds prepared by modified precipitation method. J. Mater. Sci. Mater. Electron. 2014;25:777–789. doi: 10.1007/s10856-013-5105-0. PubMed DOI

Giretova M., Medvecky L., Stulajterova R., Sopcak T., Briancin J., Tatarkova M. Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response. J. Mater. Sci. Mater. Electron. 2016;27:181. doi: 10.1007/s10856-016-5801-7. PubMed DOI

Tothova C., Mihajlovicova X., Novotny J., Nagy O., Giretova M., Kresakova L., Tomco M., Zert Z., Vilhanova Z., Varga M., et al. The Serum Protein Profile and Acute Phase Proteins in the Postoperative Period in Sheep after Induced Articular Cartilage Defect. Materials. 2019;12:142. doi: 10.3390/ma12010142. PubMed DOI PMC

Junqueira L.C.U., Bignolas G., Brentani R.R. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. J. Mol. Histol. 1979;11:447–455. doi: 10.1007/BF01002772. PubMed DOI

Rutgers M., van Pelt M., Dhert W., Creemers L., Saris D. Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage. Osteoarthr. Cartil. 2010;18:12–23. doi: 10.1016/j.joca.2009.08.009. PubMed DOI

Allen M.J., Houlton J.E.F., Adams S.B., Rushton N. The Surgical Anatomy of the Stifle Joint in Sheep. Veter Surg. 1998;27:596–605. doi: 10.1111/j.1532-950X.1998.tb00536.x. PubMed DOI

McIlwrith C.W., Frisbie D.D., Kawcak C.E., van Weeren R. The Horse. 2nd ed. Elsevier; St. Louis, MO, USA: 2016. Joint Disease; pp. 2–3.

Armiento A.R., Alini M., Stoddart M.J. Articular fibrocartilage—Why does hyaline cartilage fail to repair? Adv. Drug Deliv. Rev. 2019;146:289–305. doi: 10.1016/j.addr.2018.12.015. PubMed DOI

Gottardi R., Hansen U., Raiteri R., Loparic M., Düggelin M., Mathys D., Friederich N.F., Bruckner P., Stolz M. Supramolecular Organization of Collagen Fibrils in Healthy and Osteoarthritic Human Knee and Hip Joint Cartilage. PLoS ONE. 2016;11:e0163552. doi: 10.1371/journal.pone.0163552. PubMed DOI PMC

Guilak F., Nims R.J., Dicks A., Wu C.-L., Meulenbelt I. Osteoarthritis as a disease of the cartilage pericellular matrix. Matrix Biol. 2018;71–72:40–50. doi: 10.1016/j.matbio.2018.05.008. PubMed DOI PMC

Ravanetti F., Galli C., Manfredi E., Cantoni A.M., Scarpa E., Macaluso G.M., Cacchioli A. Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: An in vivo study. J. Negat. Results Biomed. 2015;14:2. doi: 10.1186/s12952-014-0021-5. PubMed DOI PMC

Roffi A., Kon E., Perdisa F., Fini M., Di Martino A., Parrilli A., Salamanna F., Sandri M., Sartori M., Sprio S., et al. A Composite Chitosan-Reinforced Scaffold Fails to Provide Osteochondral Regeneration. Int. J. Mol. Sci. 2019;20:2227. doi: 10.3390/ijms20092227. PubMed DOI PMC

Hoemann C.D., Guzmán-Morales J., Picard G., Chen G., Veilleux D., Chevrier A., Sim S., Garon M., Quenneville E., Lafantaisie-Favreau C.-H., et al. Guided bone marrow stimulation for articular cartilage repair through a freeze-dried chitosan microparticle approach. Materials. 2020;9:100609. doi: 10.1016/j.mtla.2020.100609. DOI

Gupta A., Bhat S., Jagdale P.R., Chaudhari B.P., Lidgren L., Gupta K.C., Kumar A. Evaluation of Three-Dimensional Chitosan-Agarose-Gelatin Cryogel Scaffold for the Repair of Subchondral Cartilage Defects: AnIn VivoStudy in a Rabbit Model. Tissue Eng. Part A. 2014;20:3101–3111. doi: 10.1089/ten.tea.2013.0702. PubMed DOI PMC

Petrovova E., Giretova M., Kvasilova A., Benada O., Danko J., Medvecky L., Sedmera D. Preclinical alternative model for analysis of porous scaffold biocompatibility in bone tissue engineering. ALTEX. 2019;36:121–130. doi: 10.14573/altex.1807241. PubMed DOI

Bell A.D., Hurtig M.B., Quenneville E., Rivard G.-É., Hoemann C.D. Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model. Cartilage. 2017;8:417–431. doi: 10.1177/1947603516676872. PubMed DOI PMC

Ruediger T., Horbert V., Reuther A., Kalla P.K., Burgkart R.H., Walther M., Kinne R.W., Mika J. Thickness of the Stifle Joint Articular Cartilage in Different Large Animal Models of Cartilage Repair and Regeneration. Cartilage. 2020;7:126–147. doi: 10.1177/1947603520976763. PubMed DOI PMC

Ahern B., Parvizi J., Boston R., Schaer T. Preclinical animal models in single site cartilage defect testing: A systematic review. Osteoarthr. Cartil. 2009;17:705–713. doi: 10.1016/j.joca.2008.11.008. PubMed DOI

Zhang L., Hu J., Athanasiou K.A. The Role of Tissue Engineering in Articular Cartilage Repair and Regeneration. Crit. Rev. Biomed. Eng. 2009;37:1–57. doi: 10.1615/CritRevBiomedEng.v37.i1-2.10. PubMed DOI PMC

Vyas C., Mishbak H., Cooper G., Peach C., Pereira R.F., Bartolo P. Biological perspectives and current biofabrication strategies in osteochondral tissue engineering. Biomanuf. Rev. 2020;5:1–24. doi: 10.1007/s40898-020-00008-y. DOI

Matsiko A., Levingstone T.J., O’Brien F.J. Advanced Strategies for Articular Cartilage Defect Repair. Materials. 2013;6:637–668. doi: 10.3390/ma6020637. PubMed DOI PMC

Eldracher M., Orth P., Cucchiarini M., Pape D., Madry H. Small Subchondral Drill Holes Improve Marrow Stimulation of Articular Cartilage Defects. Am. J. Sports Med. 2014;42:2741–2750. doi: 10.1177/0363546514547029. PubMed DOI

Pot M.W., Gonzales V.K., Buma P., IntHout J., Van Kuppevelt T.H., De Vries R.B., Daamen W.F. Improved cartilage regeneration by implantation of acellular biomaterials after bone marrow stimulation: A systematic review and meta-analysis of animal studies. PeerJ. 2016;4:e2243. doi: 10.7717/peerj.2243. PubMed DOI PMC

Fox A.J.S., Bedi A., Rodeo S.A. The Basic Science of Articular Cartilage: Structure, Composition, and Function. Sports Health A Multidiscip. Approach. 2009;1:461–468. doi: 10.1177/1941738109350438. PubMed DOI PMC

Mohan N., Mohanan P., Sabareeswaran A., Nair P. Chitosan-hyaluronic acid hydrogel for cartilage repair. Int. J. Biol. Macromol. 2017;104:1936–1945. doi: 10.1016/j.ijbiomac.2017.03.142. PubMed DOI

Novel approach for biomaterial assessment: utilizing the Ex Ovo quail cam assay for biocompatibility pre-screening

Evaluation of Angiogenesis in an Acellular Porous Biomaterial Based on Polyhydroxybutyrate and Chitosan Using the Chicken Ex Ovo Chorioallantoic Membrane Model