International Journal of Biological Macromolecules 118 (2018) 938–944 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac In vivo study on scaffolds based on chitosan, collagen, and hyaluronic acid with hydroxyapatite B. Kaczmarek a,⁎, A. Sionkowska a, M. Gołyńska b, I. Polkowska b, T. Szponder b, D. Nehrbass c, A.M. Osyczka d a Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Poland Department and Clinic of Animal Surgery, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Głęboka 30 Street, 20-612 Lublin, Poland AO Research Institute Davos (ARI), Switzerland d Department of Biology and Cell Imaging, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University in Kraków, Poland b c a r t i c l e i n f o Article history: Received 10 April 2018 Received in revised form 27 May 2018 Accepted 28 June 2018 Available online 30 June 2018 Keywords: In vitro In vivo Hydroxyapatite Scaffolds a b s t r a c t Scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with nano-hydroxyapatite were obtained with the use of the freeze-drying method. Composites swelling behavior was assessed by the liquid uptake test. The adhesion and proliferation of human osteosarcoma SaOS-2 cells on the scaffolds were examined in 4day culture. The biocompatibility of the chosen scaffolds was further studied by in vivo implantation into subcutaneous tissue of rabbits. The results showed low stability of the scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with hydroxyapatite. The addition of hydroxyapatite delayed the degradation process of the obtained scaffolds. The X-ray images of the tissues surrounding the scaffolds showed that both, the control scaffold without hydroxyapatite (HAp) and those with addition of 50% wt. HAp underwent degradation after 6 months. However, the scaffolds supplemented with 80% wt. HAp premained in the implanted place. The results showed satisfactory tissue response on the implanted scaffolds. © 2018 Elsevier B.V. All rights reserved. 1. Introduction Functional materials for biomedical application in regenerative medicine should be biocompatible and bioactive in order to create the appropriate stimulating environment for cell attachment and differentiation towards the desired cell phenotype. The implant should also be well ancored into the tissue to avoid implant loosening and adverse tissue reactions upon implant wear . Based on the above reasons, it is necessary to develop new functional materials which can easily be implanted into the human body and stay there to support the target tissue, improve their healing process, and prevent its further deterioration as a consequence of acute and chronic inﬂammation. Several natural compounds are currently examined for their biocompatibility, degradability, and non-cytotoxicity to eventually obtain materials which may ﬁnd use in tissue engineering [2–4]. Collagen and chitosan are among those extensively studied natural polymers that may ﬁnd broad range of applications in biomaterial science [5, 6]. The main goal of tissue engineering is to design implant materials that can support and stimulate damaged tissue regeneration . Several material improvements are being investigated to obtain new biocompatible substrates and scaffolds. Porous scaffolds are able to create the ⁎ Corresponding author. E-mail address: [email protected] (B. Kaczmarek). https://doi.org/10.1016/j.ijbiomac.2018.06.175 0141-8130/© 2018 Elsevier B.V. All rights reserved. appropriate environment for cells migration, proliferation, differentiation, and eventually for blood vessel invasion [8–10]. For regenerative medicine applications, any material must be characterized by the physicochemical methods followed by the in vitro and in vivo biological evaluation . Scaffolds obtained from chitosan and collagen have been previously studied  upon their implantation into ear skin and cartilage of rabbits. The immunohistochemical staining of the sections showed that the biodegrading behavior of those scaffolds was a long-termprocess. Moreover, it was found that chitosan/collagen scaffolds promoted angiogenesis . Finally, chitosan and collagen mixtures were also studied as materials potentially applicable for skin repair . These results showed that scaffolds were biocompatible and cells proliferated well on the scaffolds. Moreover, wound healing was promoted. Based on these reports, chitosan/collagen scaffolds seem good candidates for tissue engineering applications. The aim of the study was to examine the chitosan/collagen/ hyaluronic acid scaffolds supplemented with hydroxyapatite for potential tissue engineering applications. The polymeric mixture was chosen based on the previously performed miscibility studies of chitosan and collagen . The physicochemical properties of such scaffolds have already been examined [16, 17]. The hydroxyapatite addition improves mechanical and thermal properties. Moreover, calcium release was detected, which is necessary for bone calciﬁcation. In the present study, B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944 the liquid uptake ability as well as both in vitro and in vivo biological parameters were carried out to assess the scaffolds as potential candidates to apply in regenerative medicine. 939 made with 200 μm resolution. The scaffolds structure before and after immersion in PBS was compared. 2.3. Attachment and proliferation of human osteosarcoma SaOS-2 cells 2. Materials and methods 2.1. Sample preparation Collagen (Coll) was isolated from rat tail tendons . Chitosan (CTS), hyaluronic acid (HA) and nano-hydroxyapatite (particle size b 200 nm (BET); HAp) were purchased from Sigma-Aldrich company. Chitosan was of low molecular weight (DD = 77%, Mv = 5.4 × 105 g/mol ) and hyaluronic acid molecular weight was 1.8 × 106 g/mol . Collagen and chitosan solutions were prepared in 0.1 M acetic acid separately. Hyaluronic acid was dissolved in 0.1 M hydrochloric acid at 1% concentration. Chitosan and collagen were mixed in the 50/50 weight ratio. Hyaluronic acid was added in ratio 1, 2 and 5% to the chitosan and collagen. The amount of collagen, chitosan and hyaluronic acid were determined by miscibility studies . The nano-hydroxyapatite was added in ratio 50 and 80% to the polymeric mixture. After the mixture stirring the samples were placed in the sterile 24-well polystyrene plates and frozen. Samples were then lyophilized at −55 °C and 5 Pa for 48 h (ALPHA 1-2LD plus, CHRIST, Germany). 2.2. Liquid uptake test Scaffolds were immersed in 15 ml phosphate buffer solution (PBS) and incubated in 37 °C for 1 h, 1, and 3 days. After the time intervals, samples were removed. The weight of PBS before the sample immersion and after the scaffold removal was determined. The liquid uptake was then calculated: Liquid uptake ¼ mt −m0 100% m0 where mo is the scaffold weight before immersion (with dry surface) and mt after the speciﬁc immersion time. The scaffolds after the time intervals were washed with distilled water to remove PBS salt. Then, they were frozen and lyophilized. The liquid uptake analysis was repeated in triplicate. The morphology of the samples was studied using Scanning Electron Microscope (SEM) (LEO Electron Microscopy Ltd., England). The scaffolds were frozen in liquid nitrogen for 3 min and gently cut with a razor scalpel for the interior structure observation. The samples were covered with gold and scanning electron microscope images were For cell culture, scaffolds (0.5 cm height, 0.12 mm diameter) were soaked in 70% EtOH (water solution) followed by washing in sterile PBS (pH = 7.4). The scaffolds were then transferred to 24-well plates and left for 24 h to dry out. For the preliminary assessment of cell attachment and proliferation, we have chosen human osteosarcoma cell line SaOS-2. Although these cells are of tumorous origin, they are broadly used in literature for the initial assessment of biological compatibility of implant materials. Also, these cells resemble the osteoblast phenotype and respond similarly to normal human osteoblasts . Cells were seeded at the density of 15 × 104 cells/scaffold and cultured for total of 4 days in alpha-MEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. At day 1 culture, cell-seeded scaffolds were transferred to new culture plates and covered with fresh culture medium. The initial plates were examined with the CellTiter96Aqueous One Solution Cell Proliferation Assay (MTS, Promega, Poland) for any remaining cells that had not adhered to the scaffolds. Brieﬂy, MTS solution was diluted 10× in phenol-free alpha-MEM and 200-μl aliquots were added per well. The absorbance at 490 nm was measured after 30 min incubation at 37 °C in the dark. At day 4 of culture, the viability of cells was examined directly on the scaffolds using similar procedure as of day 1, except that the scaffolds were covered with 400-μl aliquots of 10× diluted MTS. This assay was successfully used by us previously to examine cell viability on polyurethane  and PLGA-bioactive glass scaffolds . Results were expressed as relative change in cell viability on HAp-modiﬁed scaffolds compared to results obtained for unmodiﬁed scaffolds. For statistical analysis, the value of p b 0.05 was considered signiﬁcant. 2.4. In vivo implant studies The study was conducted on a group of 8 male New Zealand White rabbits, at an age of X-Y weeks and a body weight (BW) ranging from 2.6 to 3.7 kg. The rabbits were purchased from the Center for Experimental Medicine at the Medical University of Silesia in Katowice. Animals were placed in the animal house for a 1-week adaptation to the new environment. All the animal surgical procedures were carried and approved by the II Local Ethical Committee for Experiments on Animals in Lublin (Agreement no. 16/2010). On the surgery day, each animal was premeditated by intramuscular injection of medetomidine at a dose of 0.5 mg/kg BW (Domitor, Orion Fig. 1. The liquid uptake of different scaffolds (chitosan/collagen mixture with 1% wt. hyaluronic acid addition – CTS/Coll/1HA, with 50% - CTS/Coll/1HA + 50%HAp respectively with 80% hydroxyapatite addition – CTS/Coll/1HA + 80%HAp, etc.). 940 B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944 The implantation site was prepared by shaving the thoracic hair in half of the ribs length on the left side of the body, at the subscapular region. The skin was disinfected with alcohol and iodine, and prepared for a sterile operation. A vertical 1 cm long cut was made with a scalpel between the 7th and 8th rib. Then, the sterile material was placed into the subcutaneous pocket. The pocket was sutured with single absorbable Pharma, Finland) and butorphanol at a dose of 0.2 mg/kg BW (Butomidor, Richter Pharma, Austria). After 10 min, an intravenous catheter was placed into the marginal vein of the ear. General anesthesia was maintained by the intravenous administration of ketamine (Bioketan, Biowet, Poland) at a dose of 30–50 mg/kg BW. The anesthesia period for each animal lasted 30 min on average. dry 1h 2 4h Fig. 2. SEM images with 200 μm resolution of a) CTS/Coll/2HA b) CTS/Coll/2HA + 80%HAp c) CTS/Coll/5HA d) CTS/Coll/5HA +80%HAp, respectively, for dry scaffold, scaffolds after 1 h and 24 h of immersion in PBS. B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944 sutures made of PGLA Lactic 4- 0 (YAVO, Poland) and the skin - with sealed with Nylon 3-0 (YAVO, Poland). The identical surgery procedures were performed on the right side of the subscapular region. The following scaffolds were implanted separately: chitosan/collagen supplemented with 2% wt. hyaluronic acid and 80% wt. hydroxyapatite (CTS/ Coll/2HA + 80%HAp), CTS/Coll supplemented with 5% wt. hyaluronic acid and 50% wt. HAp (CTS/Coll/5HA + 50%HAp) or 80% wt. HAp (CTS/Coll/5HA + 80%HAp), or unmodiﬁed scaffolds without HAp (CTS/Coll/5HA). Four rabbits (R1–R4) were implanted with CTS/Coll/ 2HA + 80% HAp on the left side and with CTS/Coll/5HA + 80%HAp on the right side. The other four rabbits (R5–R8) were implanted with CTS/Coll/5HA + 50%HAp on the left side and with CTS/Coll/5HA on the right side. After surgery, all the rabbits were individually housed in cages. In order to minimize the risk of infection, they were given enroﬂoxacin at a dose of 5 mg/kg BW IM side (Enrobioﬂox 5%, Vetoquinol, Poland). The antibiotic was administered for 3 days after surgery. The skin sutures were removed 10 days after the surgery. Six months after implantation, blood samples were taken for hematological and biochemical studies and the animals were euthanized with medetomidine at 0.5 mg/kg BW in combination with ketamine 25 mg/kg BW and ﬁnally pentobarbital (Morbital, Biowet, Poland) at a dose of 2–5 mg/kg BW. Skin samples with the subcutaneous tissue from the place where the material was initially implanted were taken for further analyses. 2.5. Hematological and biochemical parameters Blood tests included the assessment of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) amounts using a Scil Vet ABC Plus + hematology analyzer (Horiba Medical). Serum creatinine (CR) and urea nitrogen (BUN) levels as well as alanine aminotransferase (ALT) and aspartyl aminotransferase (AST) activities were assessed. The markings were made with the use of the biochemical analyzer, Mindray BS-120. The values given by Winnicka  were corrected for the above parameters. 2.6. Radiological assessment Radiological images of the harvested skin samples were taken with Planmeca Romexis, Finland and exposure parameters of 40 kV, 30 mA, 300 mAs. 2.7. Histological assessment The tissue samples (1.5 × 1.5 cm) harvested from the implant site were trimmed and then ﬁxed in a 10% buffered formalin solution. Then, they underwent standard histological processing. Sections of 5–6 μm thickness were stained with hematoxylin and eosin (HE) and examined under with a brightﬁeld light microscope. 3. Results and discussion Fig. 3. SaOs-2 cell viability at day 4 of culture comparing the different scaffolds (chitosan and collagen mixed in 50/50 weight ratio with 1% wt. hyaluronic acid addition – CTS/ Coll/1HA, with 2% - CTS/Coll/2HA, and with 5% hyaluronic acid – CTS/Coll/5HA) with 50 and 80% wt. HAp addition (*p b 0.05 vs. CTS/Coll/1HA; **p b 0.05 vs. CTS/Coll/2HA; # p b 0.05 vs. CTS/Coll/5HA; Student t-test). The scaffolds with 1% wt. HA show an increase in liquid uptake when increasing the HAp content, most pronounced at 3 d. Compared to those with 1% wt. HA, the scaffolds with 2 and 5% wt. HA content showed a decrease in liquid uptake once hydroxyapatite was added (50 or 80% wt. HAp). Note the effects of erosion, compared to the scaffolds without HAp the process starts earlier and is already visible after 5 d in the scaffolds with 80% wt. HAp. As the liquid uptake is related with the scaffolds porosity – a higher value of the liquid uptake may suggest that the scaffold presents higher porosity . The degradation of different scaffold types was detected by SEM observation of the material structure before immersion in PBS, after 1 h and 24 h (Fig. 2). The observed scaffold pores diameter was bigger when compared to that of the scaffolds with dry surface. The images show that the erosion process resulting from PBS salt presence was initiated during the immersion of all the scaffold types with and without HAp addition. 3.2. Attachment and proliferation of human osteosarcoma SaOS-2 cells Human SaOS-2 cells attached similarly to all studied scaffolds although day 1 analyses showed that the scaffolds with HAp supported cell attachment better when compared to the unmodiﬁed ones. This was based on the observation that less cells remained on the plates 1 day post-cell seeding after removing the modiﬁed scaffolds vs. unmodiﬁed ones (results not shown). Day 4 analyses (Fig. 3) showed that the addition of HAp signiﬁcantly improved cell attachment to the scaffolds and cells viability. We could actually observe that increasing Table 1 Results of blood cell count and biochemical blood tests in the study group 6 months after surgery. Rabbits 3.1. Liquid uptake tests The liquid uptake ability of the scaffold depended on its composition. The scaffolds absorbed high amounts of water, in the range of 2659–5205% after 1 h of immersion, 3168–10,463% after 3 h, and 2671–4987% after 24 h (Fig. 1). The highest liquid uptake after 1 day is related to the liquid ﬂow through the scaffold and the pores ﬁlling. After 3 days, the material degradation process caused by PBS salt has already been initiated, which is observed as liquid uptake decrease. However, the scaffolds of CTS/Coll with 1% wt. HA addition and high amount of HAp are more stable in PBS solution when compared to the scaffolds without inorganic particles. 941 R1 R2 R3 R4 R5 R6 R7 R8 Reference values Evaluated parameters WBC (109/l) RBC PLT (1012/l) (109/l) ALT (U/l) AST (U/l) CR BUN (mg/dl) (mg/dl) 14.1 12.6 10.3 11.0 8.9 11.2 7.6 7.8 2.0–15.0 8.7 8.0 7.6 7.5 6.8 7.4 8.1 8.0 4.0–9.0 43 32 38 33 38 43 44 37 31–53 48 56 74 45 50 51 50 68 42–98 70.8 189.0 146.3 208.3 107.1 91.0 166.8 201.0 44–234 698 632 586 735 574 623 716 528 120–800 18.0 19.0 17.6 24.5 21.2 17.8 14.2 14.0 13–30 Note: White blood cell count (WBC), red blood cell count (RBC), platelet level (PLT), creatinine (CR) and blood urea nitrogen (BUN) levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity evaluated for every implanted rabbit. 942 B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944 Fig. 4. The radiological evaluation of A) CTS/Coll/2HA + 80%HAp; B)CTS/Coll/5HA + 50%HAp, C) CTS/Coll/5HA + 80%HAp 6 months after surgery. Arrowhead: implanted scaffold. Fig. 5. The histological sections of the tissue surrounding A) CTS/Coll/5HA B) CTS/Coll/5HA + 50%HAp and C) CTS/Coll/5HA + 80%HAp 6 months after implantation (Scale bar indicates 100 μm (left) and with 12.6 × 20 magniﬁcation (right). T: marks the subcutismainly consisting of ﬁbrous tissue, N: nerve, A: arteriole, C: capillary, S: skeletal muscle, E: elastic ﬁbers). B. Kaczmarek et al. / International Journal of Biological Macromolecules 118 (2018) 938–944 amount of inorganic compounds in the scaffolds corresponded with increased cell attachment and viability. Similar results were presented by Jain et al. , who showed that addition of HAp to polyamide improves the MSCs cells attachment and proliferation. We have also showed that the combination of bioactive glass and HAp increases human MSC viability and promotes their osteogenic differentiation . On the other hand side, we could observe that supplementing the scaffolds with hyaluronic acid does not inﬂuence cells attachment and viability. Based on the above in vitro studies, we have chosen the following scaffolds for further in vivo analyses: chitosan and collagen mixture supplemented with 2% wt. hyaluronic acid and 80% wt. hydroxyapatite (CTS/Coll/2HA + 80%HAp); CTS/Coll enriched with 5% wt. hyaluronic acid (CTS/Coll/5HA) and 50% wt. HAp (CTS/Coll/5HA + 50%HAp) or 80% wt. HAp (CTS/Coll/5HA + 80%HAp). We assumed these scaffolds had the highest biocompatibility and could serve as potential regenerative medicine substitute. 3.3. In vivo studies 3.3.1. Macroscopic observation The post-operative wounds were healed with no complications. In the surrounding tissues, there were no inﬂammatory changes, swelling, or congestion. Upon the 6-month implantation schedule, the rabbits showed no signs of general or local health problems. 3.3.2. Hematological and biochemical parameters Hematological (WBC, RBC, PLT) and biochemical blood test results (ALP, AST, creatinine, urea nitrogen) were within the reference values (Table 1). The laboratory tests results 6 months after surgery were within the reference range for all the implanted rabbits. Animals showed no symptoms of disease or general inﬂammation associated with the implantation of the materials used. 3.3.3. Radiological assessment The results of the radiological examination are shown in Fig. 4. Six months after the materials enriched with HAp had been implanted, they were visible on X-rays. The scaffold of CTS/Coll/5HA was not captured by X-rays because it had degraded. 3.3.4. Histological assessment The implantation period allowed us to evaluate the granulation and tissue healing phase. The ﬁrst one is usually identiﬁed by the presence of angioblasts, ﬁbroblasts, and collagen synthesis . The images (Fig. 5) allow to observe the physiological structures of the rabbit skin as lamina subcutanea with elastic and collagen ﬁbers and nerves. The scaffold made of chitosan and collagen with 5% wt. hyaluronic acid addition biodegraded because of its low stability. In contrast, scaffolds with addition of HAp were still visible at the implantation place after 6 months. In the scaffold, the ﬁbroblasts presence was noticed. They grew into the scaffolds with the HAp addition (Fig. 5b and c). The scaffold morphology was similar to the surrounding tissue . For the scaffolds with HAp, the formation of blood vessels (capillaries) was observed, which is essential for tissue regeneration. Moreover, the vascularization was observed as physiologic for subcutis consisting of mid-sized vessels often in pairs as well in trias with nerve. The results showed that scaffolds based solely on natural polymers such as chitosan, collagen, and hyaluronic acid undergo degradation and the HAp presence changes the long-term stability. Summarized, the analyses of cell and tissue responses to the studied materials showed no inﬂammation at the implantation sites. The implants did not elicit any adverse or unexpected reactions at the adjacent soft tissues. Many reports have indicated the accelerating effects of chitosan and its derivatives on wound healing . Because of its antimicrobial properties, chitosan has been widely investigated as an antimicrobial agent 943 used for preventing infections, which is advantageous in the case of a substance used as an implant component . Host reactions following biomaterials implantation include injury, blood-material interactions, acute or chronic inﬂammation, and granulation tissue development as a reaction of the host to foreign body. Depending on the extent of an injury, the acute inﬂammatory response to biomaterials at the implant site usually occurs quickly, within a few days. Then, chronic inﬂammation characterized by the presence of mononuclear cells (i.e. monocytes and lymphocytes) at the implant site develops. The presence of chronic inﬂammatory responses for a period longer than 3 weeks usually indicates an infection. After the chronic inﬂammation stage, new healthy tissue can be identiﬁed, the granulation tissue characterized by macrophages presence, ﬁbroblasts inﬁltration, and neovascularization . 4. Conclusions In this study, we veriﬁed the biocompatibility of chitosan, collagen, and hyaluronic acid-based scaffolds enriched with nanohydroxyapatite. 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