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Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect Yingjie Wang a, 1, Wei Zhu a, 1, Ke Xiao a, Zeng Li a, Qi Ma a, Weifeng Li a, Songpo Shen b, Xisheng Weng a, * a Department of Orthopaedics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China b Department of Orthopaedics, Beijing Tongren Hospital, The Affiliated Hospital of Capital University of Medical Sciences, Beijing 100730, China
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Article history: Received 24 October 2018 Received in revised form 9 November 2018 Accepted 15 November 2018 Available online xxx
Background: HA modified by bisphosphonate (BP) (HA-BP) was synthesized by chemical reaction and possessed promising properties such as self-healing, injection ability, and strong adhesion. The main aim of this study was to confirm its role in promoting osteogenic differentiation in vitro and bone regeneration in vivo. Methods: The cell biocompatibility of this material was determined using the CCK-8 assay. Alkaline phosphatase (ALP), osteocalcin (OT), vascular endothelial growth factor (VEGF), and collagen I were assessed by quantitative real-time polymerase chain reaction (Q-PCR) in the treated group. The number and density of calcium nodules and ALP were evaluated by Alizarin Red staining and ALP staining. We have successfully developed an animal model simulating osteonecrosis of the femoral head (ONFH). Utilizing this animal model, the impact of HA-BP/CaP on bone formation was assessed. The amount of bone regeneration at 1 and 2 months after HA-BP/CaP injection was estimated by micro-computed tomography (micro-CT) analysis and H&E, collagen I, and periostin staining. Results: The number of cells gradually increased in the experimental group over time and was close to that of the blank control group. ALP, collagen I, and VEGF expression was significantly higher in the experimental group than in the blank group (VEGF, ALP, both **p < 0.01; collagen I, ***p<0.001). In addition, the number and density of calcium nodules and ALP was clearly greater in the material group than in the control group. The quantification analysis showed that the mineral contents of regenerated bone at 1 and 2 months after HA-BP/CaP injection were significantly greater than those in the control group, according to microCT evaluation (**p<0.01). The amount of organic components in the HA-BP/CaP group was greater than that in the control group after decalcification and H&E staining. In addition, collagen I and periostin staining further confirmed the results of H&E staining. Conclusion: This material can boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. It can intensely accelerate bone regeneration in vivo, which is a promising strategy for tissue engineering. © 2018 Elsevier Inc. All rights reserved.
Keywords: HA-BP/CaP Femoral head necrosis Bone regeneration
1. Introduction Osteonecrosis of the femoral head (ONFH) is a severely disabling disease with various etiologies such as glucocorticoid use, alcohol and trauma. However, its underlying mechanism is not fully
* Corresponding author. E-mail address: [email protected] (X. Weng). 1 Contributed equally to this work.
understood [1,2]. In America, about 20,000 to 30,000 people are diagnosed with ONFH per year [3]. The incidence of non-traumatic ONFH is 1.91/100,000/year [4]. Moreover, the incidence of steroidinduced ONFH was 9e40% in patient populations receiving longterm treatment. ONFH mainly affects middle-aged and young people [5]. The natural course of ONFH is osteonecrosis, followed by collapse and osteoarthritis of the femoral head and hip joint, respectively. In the case of collapse, osteonecrosis and total hip replacement are necessary [6]. Therefore, it is extremely important
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Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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to implement early, effective, and efficient interventions to prevent the need for surgery. In ONFH, non-articular replacement therapy is the main approach to prevent collapse [7]. The early treatment for ONFH mainly involves core decompression to remove the necrotic bone tissue, and then, autologous or allogeneic bone is used to fill in the bone defect. Unfortunately, autologous bone grafts have disadvantages such as less bone mass; requirement of additional surgeries; longer operation times; increased blood loss and related complications; hospitalization time; and medical expenses [8e10]. Animal models of skull defects in healthy bone are the most common, but to increase their clinical relevance, it is urgent to successfully develop a novel animal model simulating ONFH. There are two vital challenges: establishing both an animal model of ONFH and a safe and effective biological material that promotes bone regeneration at the site of ONFH. 2. Experimental section 2.1. Materials Bisphosphonate-modified hyaluronic acid (HA-BP/Ca P) was kindly provided by Dmitri Ossipov and Liyang Shi. The synthesis of HA-BP/Ca P was performed following a previously reported procedure [11]. 2.2. Safety and effectiveness of HA-BP/CaP in vivo and in vitro 2.2.1. Cell cytotoxicity and proliferation Mouse embryonic osteogenic precursor cells (MC3T3-E1) purchased from the National Infrastructure of Cell Line Resource were employed to estimate the biocompatibility of this biomaterial. First, 200 vital challenges: est(containing 10,000 cells) was seeded into the lower chamber of a 24-well Corning Transwell plate (0.4 mm pore diameter), and then, 800 ml complete medium (CM) was added, which was composed of Dulbecco's modified Eagle's medium (DMEM H-21 4.5 g/L glucose), 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) bi-antibiotic (penicillin and streptomycin). Before placing 0.02 ml of the hybrid hydrogel onto the upper chamber, we waited 12 h to allow the cells sufficient time to adhere. There were 3 parallel wells utilized for both groups to reduce random errors. The cells and materials were co-cultured in an atmosphere of 37 C, 5% carbon dioxide, and a relative humidity of 95%. The control group consisted of cells alone. The time point of placing materials was marked as day 0. The cell viability was determined using a Cell Counting Kit-8 (CCK-8) kit purchased from Dojindo (Japan). On days 1, 3, 5, and 7, after the upper chambers were removed, the medium was totally discarded, and then a 1-ml solution was added consisting of 100 ml CCK-8 and 900 ml CM into the lower chamber. The reaction system was co-cultured for 2 h in a cell culture incubator. After co-culture, 100 ml of the test solution was added to a 96-well plate in three different parts, respectively, of each well. The optical density values at 450 ± 5 nm were detected using a microplate reader. DMEM, FBS, and bi-antibiotic were purchased from Gibco (USA). The 24-well Transwell plates and 96well plates were purchased from Corning (USA). The multifunctional full-wavelength microplate reader was purchased from Thermo (USA). 2.2.2. Real-time PCR for markers of osteogenic differentiation MC3T3-E1 cells were used to assess the role of HA-BP/CaP in osteogenic differentiation in vitro. The method of co-culture is described above. Fresh CM was substituted for former medium every other day. After 14 days, each dish was washed twice with phosphate buffered saline (PBS), and total RNA was extracted from
the cells. Quantitative real-time polymerase chain reaction (Q-PCR) was performed to compare the gene transcription levels of alkaline phosphatase (ALP), osteocalcin (OT), vascular endothelial growth factor (VEGF), collagen I, and GAPDH, which served as a housekeeping gene. The PBS and RNA extraction kit were purchased from Gbico (USA). Primers were designed and synthesized by Servicebio (China). The quantitative PCR instrument was purchased from ABI (USA), model 7500 Fast DX. 2.2.3. Osteogenic differentiation in vitro For determining the role of this biomaterial in facilitating osteogenic differentiation of MC3E3-E1 cells and further visually observing these changes, the following protocol was designed: (1) experimental group: osteogenic differentiation medium (ODM) combined with HA-BP/CaP to induce MC3E3-E1 cell differentiation; (2): control group: only ODM to induce MC3E3-E1 cell differentiation. The protocol of co-culture was almost the same as that for the biocompatibility test. Assessments were carried out after 7 days, including Alizarin Red staining (staining calcium salt) and ALP staining. ODM was purchased from Cyagen Bioscience (USA), ALP kits were purchased from Beyotime (China), and Alizarin Red was purchased from Solarbio (USA). 2.3. New Zealand white rabbit ONFH model for bone regeneration assessment in vivo 2.3.1. Animals This experiment was performed according to the National Institutes of Health guidelines for the use of experimental animals. This protocol was permitted by the Animal Care and Use Committee of Peking Union Medical College Hospital. In total, 20 healthy, skeletally mature female New Zealand white rabbits were purchased from the animal house of Peking Union Medical College Hospital, with an average weight of 2.5 kg (ranging from 2.3 to 2.7 kg). All rabbits were housed and bred in a temperature-controlled room (25 C), with a relative humidity of 40e60%, and a 12-h light and 12-h dark cycle. Moreover, all rabbits had free access to food and water under a standard chew diet. After allowing one week to adapt to the new surroundings, all animals were accurately weighed and randomly divided into two equal-sized independent groups: treated and untreated groups, with each group consisting of 10 rabbits. 2.3.2. Surgical procedure All 20 rabbits were anaesthetized by injecting 3% pentobarbital (1 ml/kg) into the ear vein and then fixed onto the operating table. After the operative region was shaved and sterilized, the skin around the proximal femur on the left hindlimb, the subcutaneous tissue, and muscle space were cut open one by one to expose the bone marks of the greater trochanter and the shaft of the femur. With a Kirschner wire (K-wire), a tunnel was drilled out from the mark to the femoral head. The specific parameters are described (Fig. 1). The end of the K-wire was soaked in liquid nitrogen for more than 10 s, and then the K-wire was fully inserted into the tunnel for more than 10 s. The freezing steps were repeated 5 times. Then, 0.2 ml of the hybrid hydrogel was injected into the area of the bone defect for each rabbit in the experimental group, while the control rabbits were injected with 0.2 ml saline. A total of 10 rabbits were implanted with the hybrid hydrogel, and the remaining 10 animals were injected with saline. To prevent infection, at the time of surgery and the day after, all rabbits were intramuscularly injected with 200,000 U penicillin. 2.3.3. Evaluation of ONFH The femurs of other 10 rabbits were harvested and soaked in
Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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Fig. 1. The details regarding the standard animal model of ONFH are as follows: the angle between the K-wire and femur shaft was almost 45 , and the length between the great trochanter and the mark was about 6 mm. After adjusting the angle and distance, the K-wire was slightly skewed to the right. The depth of the tunnel was 25e30 mm.
neutral formalin at 1 month after the surgery. Gross observation, micro-CT, MRI, and H&E staining analysis were used to assess the ONFH. 2.3.4. Micro-CT evaluation and immunohistochemical analysis The femurs were harvested and soaked in neutral formalin at 1 and 2 months after the surgery. The femoral heads of the rabbits were scanned via micro-computed tomography (micro-CT) to measure the bone volume using the tissue with signals ranging from 300 to 900 HU. The micro-CT system was purchased from Siemens (Germany), and the resolution was 10.3 mm. Then, the femur samples were decalcified, embedded in paraffin, and the targeted areas were sectioned to 50 um thickness by a microtome. Finally, these tissue sections were stained with hematoxylin and eosin (H&E), Sirius red, and periostin antibody to detect bone formation via microscopy. Rabbit anti-periostin/HRP (bs-4994R-HRP) and HRP-goat anti-rabbit IgG (074-15-06) were purchased from Bioss (China) and KPL (U.S.), respectively. 2.4. Statistical analysis All test results are expressed in groups in terms of ALP, OT, VEGF, and collagen I levels, and relevant indicators about bone volumes of the femoral head defect area were compared using Student's t-test after the homogeneity of variance was confirmed. Statistical
Fig. 2. Establishment of ONFH in New Zealand white rabbits at 1 month after surgery. ONFH was observed in the experiment group as measured by micro-CT analysis: B-C) sagittal cross-section views, magnetic resonance imaging: D-E) sagittal cross-section views, H&E staining: F-G. A: The subchondral bone of the femoral head was dark red, and the black arrow shows osteonecrosis. B: untreated group. C: treated group, the blue arrow points to the area of ONFH. D: untreated group. E: treated group, the blue arrow points to the area of ONFH. F: untreated group. G: treated group, almost no stained nuclei were observed (black arrow).
analyses were performed by SPSS 21.0 software. P<0.05 was defined as a statistically significant difference. All data are represented as means ± SD (standard deviation). 3. Results 3.1. The establishment of the animal model for ONFH The subchondral bone of the femoral head in the experimental group presented the establishment of the ONFH animal model because of the dark red area on the surface of the femoral head, while the untreated group appeared normal (Fig. 2A). To confirm the model, micro-CT and MRI analysis were employed to detect the specific site. Compared to the untreated group (Fig. 2B), in the treated group, the trabecula of the subchondral bone was disordered and sparse and the outermost bone and the osteoepiphysis
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were thin (Fig. 2C). In addition, the femoral head of the treated group (Fig. 2E) showed irregular high-and low-mixed signal areas, which indicates the occurrence of ONFH, and bone signals in the untreated group were uniform (Fig. 2D). The results of H&E staining (40 times magnification) showed that the bone sag became empty, and almost no stained nuclei were observed (Fig. 2G). Conversely, the stroma and nuclei were evenly stained, and there was no nucleus pyknosis in the untreated group (Fig. 2F). 3.2. Self-healing hydrogel supports MC3T3-E1 growth The number and viability of MC3T3-E1 cells was determined using CCK-8 reagent on days 1, 3, 5, and 7. The optical density value positively correlated with the cell number. Therefore, the number of cells gradually increased in the experimental group over time and was close to that of the blank control group (Fig. 3A). The result clearly illustrated that this composite possesses good biocompatibility. 3.3. In vitro osteogenic potential of self-healing hydrogel Other than the biosafety of the gel, its biological effectiveness is of great importance. A series of osteogenic differentiation indicators were assessed. As shown in Fig. 3B, the expression levels of every indicator in the material group were significantly higher than those in the blank group, with a statistically significant difference. The results of the ALP and Alizarin Red staining were promising. The number and density of calcium nodules in the material group was clearly increased compared to the control group (Fig. 3CeF). The area of stained ALP in the experimental group was significantly greater than that in the control group. All the experimental results above indicate that the hybrid hydrogel can support the growth of MC3T3-E1 cells and also promote the osteogenic differentiation of MC3T3-E1 cells in vitro. 3.4. Bone regeneration facilitated by implantation of injectable hydrogel 3.4.1. Micro-CT evaluation Fig. 4 shows that the amount of bone regeneration at 1 and 2 months after injection of HA-BP/CaP in the experimental group was significantly greater than that in the control group. The amount of regenerated bone at 2 months after surgery was significantly greater than that at 1 month post-surgery (Fig. 4AeE).
Fig. 4. A-D: 3D reconstruction views, A corresponds to 1 month after surgery without hydrogel placement; B to 1 month after hydrogel implantation; C corresponds to 2 months after surgery without hydrogel placement; D to 2 months after hydrogel implantation. E: Quantification of the newly formed bone volume per osteonecrosis area. H&E after hydrogel implantation for 1 month (G) and 2 months (I), without hydrogel for 1 month (F) and 2 months (H). Black arrows: collagen, white arrows: osteoblast cells. The scale bar is 100 after hydrogel implantation for 1 month (was perioK) and 2 (M) months, without hydrogel for 1 month (J) and 2 months (L). More swirling bone units were easily found in the implanted group (K, M). White arrows: collagen I. The scale bar is 50 he implanted group r is 100 after hydrogel implantation for 1 moO) and 2 (Q) months, respectively. Black arrows: periostin, red arrows: collagen.
3.4.2. Histological and immunohistochemical analysis It was observed that at 1 and 2 months, the amount of regenerated collagen and the number of osteoblasts in the HA-BP/CaP group were higher than in the control group after decalcification and H&E staining. Collagen content in the material group at 2 months was significantly higher than that at 1 month and was the same as that in the control group (Fig. 4FeI). After staining with Sirius Red and observation by polarized light microscopy, a large quantity of newly regenerated type I collagen was observed (type I collagen fibers are closely arranged, showing a strong birefringence and yellow, orange, and red coarse fibers), which further verifies the results of the H&E staining (Fig. 4J-M). Through immunohistochemical staining, it was observed that the hyperchromatic linear region around the bone trabeculae was periostin, and the expression of periostin at 1 and 2 months in the experimental group was significantly higher than that in the control group. The expression level in the experimental group at 2 months was higher than that at 1 month and was the same as that in the control group (Fig. 4NeQ). 4. Discussion
Fig. 3. The biocompatibility and role in promoting osteogenesis differentiation of HABP/CaP. A: CCK-8 results for biocompatibility. B: The expression of ALP, OT, collagen I, and VEGF in MC3T3-E1 cells on the hybrid hydrogel on day 14. C/D: white arrow: calcium nodules, ODM: osteogenic differentiation medium. E/F: white arrow: the sites of alkaline phosphatase. *p < 0.05, **p < 0.01, ***p < 0.001.
A non-covalent crosslinked hybrid hydrogel with self-healing, shear-thinning, and strong adhesion properties was successfully synthesized [11]. Additionally, this material could boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. Once injected into the site of ONFH in a clinically relevant animal model, it could intensely accelerate bone regeneration. Using an ONFH
Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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animal model, we confirmed that this biomaterial can quickly promote bone regeneration in the osteonecrotic area other than healthy bone defect. This finding is promising because it could be an ideal strategy for certain ONFH patients who need to undergo a total hip arthroplasty in the next few years. In recent years, more self-healing gels have been synthesized based on the biocompatibility of HA. However, there are relatively few studies on the use of gels [12]. HA-based composites formed by irreversible covalent bonds exist but unfortunately are not injectable or self-healing [13] and are rarely applied in the field of bone regeneration [14]. Although the focus of few researchers has been on bone or cartilage regeneration [15], one of the critical drawbacks in these studies is that the materials were applied to healthy bone or cartilage defects. The animal models are inconsistent with the pathophysiological mechanisms observed in patients, especially in ONFH. In the present study, the New Zealand rabbit model of ONFH was produced. Clinical core decompression surgery and bone grafting were simulated in the animal model, and bone grafts were replaced with the HA-BP/CaP. This material has significant advantages in clinical use: simpler operation, small wound on the skin and the surrounding soft tissues, shorter operation time, and matching of the shape of the injected hydrogel to that of the bone defect area [16e18]. The conditions for bone regeneration are determined by the interaction between the cells in the bone defect area and the surface of the implant materials. Once the hybrid hydrogel is implanted into the animal, the immune system will recognize the antigenic determinant presented on the HA-based composite, and then, the immune system will probably reject the implant. Hence, its use will not facilitate bone regeneration on account of immunological rejection. Accordingly, one of the keys to success in this approach is reducing the immunological rejection and increasing the biocompatibility of the biomaterials [19]. The results of the CCK-8 assay proved the excellent compatibility of HA-BP/CaP. In addition to the biosafety of the gel, its biological effectiveness is of great importance. A series of osteogenic differentiation indicators were assessed. ALP, OT, collagen I, and VEGF contents were estimated by Q-PCR. In addition, ALP and Alizarin Red staining were used to evaluate the calcium nodules and the amount of ALP. All the experimental results showed that the hybrid hydrogel can support the growth of MC3T3-E1 cells and also promote the osteogenic differentiation of MC3T3-E1 cells in vitro. HA-BP/CaP contains a large number of proteins, with a threedimensional structure that can mimic the extracellular matrix [20]. Calcium phosphonate, a natural component of bone, possesses osteoinductivity and osteoconductivity [21]. BPs are widely used clinically in the treatment of osteoporosis and are also incorporated as bioactive molecules into a variety of bio-scaffolds to promote bone regeneration [22]. BPs have the property of targeted binding of bone minerals. Therefore, we predicted that the material can enhance tissue regeneration. Based on the animal model, New Zealand white rabbits were treated according to the procedure that is applied in clinical practice to treat patients with ONFH [23]. Bone regeneration was assessed by micro-CT and histology. Compared to our reported biomaterial (Am-HA-BP.CaP@mSF) [24], HA-BP/CaP exhibits excellent biological properties and its osteogenic differentiation and bone regeneration promoting abilities are similar to those of Am-HA-BPnatural component of bone, possesses osteoinductivity and osteoconductivitye HA-BP/CaP was applied to an osteonecrotic bone area. Additionally, more tests were performed to confirm new bone formation by CT, collagen by H&E staining, collagen I by Sirius red staining, and periostin by immunohistochemistry. The most abundant organic component in bone is collagen I [25]. The periostin in bone tissue is mainly expressed in
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the periosteum. It controls many stages of intramembranous osteogenesis by facilitating osteoblast cell adhesion, proliferation, and differentiation [26,27]. To determine the impact of HA-BP/CaP on osteogenic differentiation in vitro, Q-PCR was used and extracellular matrix mineralization indicators, such as calcium nodules and ALP, were assessed. Interestingly, all results illustrated the excellent biological effectiveness of ONFH in promoting osteogenic differentiation in vitro and bone regeneration in vivo. 5. Conclusion A non-covalent crosslinked hybrid hydrogel with self-healing, shear-thinning, and strong adhesion properties was successfully synthesized. Additionally, this material can boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. Once injected into the site of ONFH in the novel, clinically relevant animal model, it can intensely accelerate bone regeneration and is a promising tissue engineering strategy. In the future, other bioactive molecules may be loaded onto the biomaterial to further improve the performance of this material, such as VEGF and bone morphogenetic protein-2. Conflicts of interest The authors declare that they have no competing interests. Acknowledgments We acknowledge financial support from 1) Beijing natural science foundation youth project NO.7184235; 2) China postdoctoral foundation NO.62 general program. References [1] K.J. Guo, et al., The influence of age, gender and treatment with steroids on the incidence of osteonecrosis of the femoral head during the management of severe acute respiratory syndrome: a retrospective study, Bone Joint Lett. J 96b (2) (2014) 259e262. [2] A. Wang, M. Ren, J. Wang, The Pathogenesis of Steroid-induced Osteonecrosis of the Femoral Head: a Systematic Review of the Literature, Gene, 2018. [3] J.R. Lieberman, et al., Osteonecrosis of the hip: management in the 21st century, Instr. Course Lect. 52 (2003) 337e355. [4] K. Ikeuchi, et al., Epidemiology of nontraumatic osteonecrosis of the femoral head in Japan, Mod. Rheumatol. 25 (2) (2015) 278e281. [5] P. Luo, et al., The role of autophagy in steroid necrosis of the femoral head: a comprehensive research review, Int. Orthop. 42 (7) (2018) 1747e1753. [6] J.S. Kang, et al., The natural history of asymptomatic osteonecrosis of the femoral head, Int. Orthop. 37 (3) (2013) 379e384. [7] D. van der Jagt, et al., Osteonecrosis of the femoral head: evaluation and treatment, J. Am. Acad. Orthop. Surg. 23 (2) (2015) 69e70. [8] Y.A. Fillingham, B.A. Lenart, S. Gitelis, Function after injection of benign bone lesions with a bioceramic, Clin. Orthop. Relat. Res. 470 (7) (2012) 2014e2020. [9] C.G. Finkemeier, Bone-grafting and bone-graft substitutes, J Bone Joint Surg Am 84-a (3) (2002) 454e464. [10] M. Putzier, et al., Allogenic versus autologous cancellous bone in lumbar segmental spondylodesis: a randomized prospective study, Eur. Spine J. 18 (5) (2009) 687e695. [11] M.R. Nejadnik, et al., Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles, Biomaterials 35 (25) (2014) 6918e6929. [12] Q. Wang, et al., Injectable PLGA based colloidal gels for zero-order dexamethasone release in cranial defects, Biomaterials 31 (18) (2010) 4980e4986. [13] B. Rybtchinski, Adaptive supramolecular nanomaterials based on strong noncovalent interactions, ACS Nano 5 (9) (2011) 6791e6818. [14] Q.G. Wang, et al., Molecular profiling of single cells in response to mechanical force: comparison of chondrocytes, chondrons and encapsulated chondrocytes, Biomaterials 31 (7) (2010) 1619e1625. [15] Hou, S., et al., Rapid self-integrating, injectable hydrogel for tissue complex regeneration. Adv Healthc Mater, 2015. 4(10): p. 1491-1495, 1423. [16] P. Ni, et al., Injectable thermosensitive PEG-PCL-PEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects, Biomaterials 35 (1) (2014) 236e248. [17] K. Bergman, et al., Injectable cell-free template for bone-tissue formation, J. Biomed. Mater. Res. 91 (4) (2009) 1111e1118.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect Yingjie Wang a, 1, Wei Zhu a, 1, Ke Xiao a, Zeng Li a, Qi Ma a, Weifeng Li a, Songpo Shen b, Xisheng Weng a, * a Department of Orthopaedics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China b Department of Orthopaedics, Beijing Tongren Hospital, The Affiliated Hospital of Capital University of Medical Sciences, Beijing 100730, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2018 Received in revised form 9 November 2018 Accepted 15 November 2018 Available online xxx
Background: HA modified by bisphosphonate (BP) (HA-BP) was synthesized by chemical reaction and possessed promising properties such as self-healing, injection ability, and strong adhesion. The main aim of this study was to confirm its role in promoting osteogenic differentiation in vitro and bone regeneration in vivo. Methods: The cell biocompatibility of this material was determined using the CCK-8 assay. Alkaline phosphatase (ALP), osteocalcin (OT), vascular endothelial growth factor (VEGF), and collagen I were assessed by quantitative real-time polymerase chain reaction (Q-PCR) in the treated group. The number and density of calcium nodules and ALP were evaluated by Alizarin Red staining and ALP staining. We have successfully developed an animal model simulating osteonecrosis of the femoral head (ONFH). Utilizing this animal model, the impact of HA-BP/CaP on bone formation was assessed. The amount of bone regeneration at 1 and 2 months after HA-BP/CaP injection was estimated by micro-computed tomography (micro-CT) analysis and H&E, collagen I, and periostin staining. Results: The number of cells gradually increased in the experimental group over time and was close to that of the blank control group. ALP, collagen I, and VEGF expression was significantly higher in the experimental group than in the blank group (VEGF, ALP, both **p < 0.01; collagen I, ***p<0.001). In addition, the number and density of calcium nodules and ALP was clearly greater in the material group than in the control group. The quantification analysis showed that the mineral contents of regenerated bone at 1 and 2 months after HA-BP/CaP injection were significantly greater than those in the control group, according to microCT evaluation (**p<0.01). The amount of organic components in the HA-BP/CaP group was greater than that in the control group after decalcification and H&E staining. In addition, collagen I and periostin staining further confirmed the results of H&E staining. Conclusion: This material can boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. It can intensely accelerate bone regeneration in vivo, which is a promising strategy for tissue engineering. © 2018 Elsevier Inc. All rights reserved.
Keywords: HA-BP/CaP Femoral head necrosis Bone regeneration
1. Introduction Osteonecrosis of the femoral head (ONFH) is a severely disabling disease with various etiologies such as glucocorticoid use, alcohol and trauma. However, its underlying mechanism is not fully
* Corresponding author. E-mail address: [email protected] (X. Weng). 1 Contributed equally to this work.
understood [1,2]. In America, about 20,000 to 30,000 people are diagnosed with ONFH per year [3]. The incidence of non-traumatic ONFH is 1.91/100,000/year [4]. Moreover, the incidence of steroidinduced ONFH was 9e40% in patient populations receiving longterm treatment. ONFH mainly affects middle-aged and young people [5]. The natural course of ONFH is osteonecrosis, followed by collapse and osteoarthritis of the femoral head and hip joint, respectively. In the case of collapse, osteonecrosis and total hip replacement are necessary [6]. Therefore, it is extremely important
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Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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to implement early, effective, and efficient interventions to prevent the need for surgery. In ONFH, non-articular replacement therapy is the main approach to prevent collapse [7]. The early treatment for ONFH mainly involves core decompression to remove the necrotic bone tissue, and then, autologous or allogeneic bone is used to fill in the bone defect. Unfortunately, autologous bone grafts have disadvantages such as less bone mass; requirement of additional surgeries; longer operation times; increased blood loss and related complications; hospitalization time; and medical expenses [8e10]. Animal models of skull defects in healthy bone are the most common, but to increase their clinical relevance, it is urgent to successfully develop a novel animal model simulating ONFH. There are two vital challenges: establishing both an animal model of ONFH and a safe and effective biological material that promotes bone regeneration at the site of ONFH. 2. Experimental section 2.1. Materials Bisphosphonate-modified hyaluronic acid (HA-BP/Ca P) was kindly provided by Dmitri Ossipov and Liyang Shi. The synthesis of HA-BP/Ca P was performed following a previously reported procedure [11]. 2.2. Safety and effectiveness of HA-BP/CaP in vivo and in vitro 2.2.1. Cell cytotoxicity and proliferation Mouse embryonic osteogenic precursor cells (MC3T3-E1) purchased from the National Infrastructure of Cell Line Resource were employed to estimate the biocompatibility of this biomaterial. First, 200 vital challenges: est(containing 10,000 cells) was seeded into the lower chamber of a 24-well Corning Transwell plate (0.4 mm pore diameter), and then, 800 ml complete medium (CM) was added, which was composed of Dulbecco's modified Eagle's medium (DMEM H-21 4.5 g/L glucose), 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) bi-antibiotic (penicillin and streptomycin). Before placing 0.02 ml of the hybrid hydrogel onto the upper chamber, we waited 12 h to allow the cells sufficient time to adhere. There were 3 parallel wells utilized for both groups to reduce random errors. The cells and materials were co-cultured in an atmosphere of 37 C, 5% carbon dioxide, and a relative humidity of 95%. The control group consisted of cells alone. The time point of placing materials was marked as day 0. The cell viability was determined using a Cell Counting Kit-8 (CCK-8) kit purchased from Dojindo (Japan). On days 1, 3, 5, and 7, after the upper chambers were removed, the medium was totally discarded, and then a 1-ml solution was added consisting of 100 ml CCK-8 and 900 ml CM into the lower chamber. The reaction system was co-cultured for 2 h in a cell culture incubator. After co-culture, 100 ml of the test solution was added to a 96-well plate in three different parts, respectively, of each well. The optical density values at 450 ± 5 nm were detected using a microplate reader. DMEM, FBS, and bi-antibiotic were purchased from Gibco (USA). The 24-well Transwell plates and 96well plates were purchased from Corning (USA). The multifunctional full-wavelength microplate reader was purchased from Thermo (USA). 2.2.2. Real-time PCR for markers of osteogenic differentiation MC3T3-E1 cells were used to assess the role of HA-BP/CaP in osteogenic differentiation in vitro. The method of co-culture is described above. Fresh CM was substituted for former medium every other day. After 14 days, each dish was washed twice with phosphate buffered saline (PBS), and total RNA was extracted from
the cells. Quantitative real-time polymerase chain reaction (Q-PCR) was performed to compare the gene transcription levels of alkaline phosphatase (ALP), osteocalcin (OT), vascular endothelial growth factor (VEGF), collagen I, and GAPDH, which served as a housekeeping gene. The PBS and RNA extraction kit were purchased from Gbico (USA). Primers were designed and synthesized by Servicebio (China). The quantitative PCR instrument was purchased from ABI (USA), model 7500 Fast DX. 2.2.3. Osteogenic differentiation in vitro For determining the role of this biomaterial in facilitating osteogenic differentiation of MC3E3-E1 cells and further visually observing these changes, the following protocol was designed: (1) experimental group: osteogenic differentiation medium (ODM) combined with HA-BP/CaP to induce MC3E3-E1 cell differentiation; (2): control group: only ODM to induce MC3E3-E1 cell differentiation. The protocol of co-culture was almost the same as that for the biocompatibility test. Assessments were carried out after 7 days, including Alizarin Red staining (staining calcium salt) and ALP staining. ODM was purchased from Cyagen Bioscience (USA), ALP kits were purchased from Beyotime (China), and Alizarin Red was purchased from Solarbio (USA). 2.3. New Zealand white rabbit ONFH model for bone regeneration assessment in vivo 2.3.1. Animals This experiment was performed according to the National Institutes of Health guidelines for the use of experimental animals. This protocol was permitted by the Animal Care and Use Committee of Peking Union Medical College Hospital. In total, 20 healthy, skeletally mature female New Zealand white rabbits were purchased from the animal house of Peking Union Medical College Hospital, with an average weight of 2.5 kg (ranging from 2.3 to 2.7 kg). All rabbits were housed and bred in a temperature-controlled room (25 C), with a relative humidity of 40e60%, and a 12-h light and 12-h dark cycle. Moreover, all rabbits had free access to food and water under a standard chew diet. After allowing one week to adapt to the new surroundings, all animals were accurately weighed and randomly divided into two equal-sized independent groups: treated and untreated groups, with each group consisting of 10 rabbits. 2.3.2. Surgical procedure All 20 rabbits were anaesthetized by injecting 3% pentobarbital (1 ml/kg) into the ear vein and then fixed onto the operating table. After the operative region was shaved and sterilized, the skin around the proximal femur on the left hindlimb, the subcutaneous tissue, and muscle space were cut open one by one to expose the bone marks of the greater trochanter and the shaft of the femur. With a Kirschner wire (K-wire), a tunnel was drilled out from the mark to the femoral head. The specific parameters are described (Fig. 1). The end of the K-wire was soaked in liquid nitrogen for more than 10 s, and then the K-wire was fully inserted into the tunnel for more than 10 s. The freezing steps were repeated 5 times. Then, 0.2 ml of the hybrid hydrogel was injected into the area of the bone defect for each rabbit in the experimental group, while the control rabbits were injected with 0.2 ml saline. A total of 10 rabbits were implanted with the hybrid hydrogel, and the remaining 10 animals were injected with saline. To prevent infection, at the time of surgery and the day after, all rabbits were intramuscularly injected with 200,000 U penicillin. 2.3.3. Evaluation of ONFH The femurs of other 10 rabbits were harvested and soaked in
Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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Fig. 1. The details regarding the standard animal model of ONFH are as follows: the angle between the K-wire and femur shaft was almost 45 , and the length between the great trochanter and the mark was about 6 mm. After adjusting the angle and distance, the K-wire was slightly skewed to the right. The depth of the tunnel was 25e30 mm.
neutral formalin at 1 month after the surgery. Gross observation, micro-CT, MRI, and H&E staining analysis were used to assess the ONFH. 2.3.4. Micro-CT evaluation and immunohistochemical analysis The femurs were harvested and soaked in neutral formalin at 1 and 2 months after the surgery. The femoral heads of the rabbits were scanned via micro-computed tomography (micro-CT) to measure the bone volume using the tissue with signals ranging from 300 to 900 HU. The micro-CT system was purchased from Siemens (Germany), and the resolution was 10.3 mm. Then, the femur samples were decalcified, embedded in paraffin, and the targeted areas were sectioned to 50 um thickness by a microtome. Finally, these tissue sections were stained with hematoxylin and eosin (H&E), Sirius red, and periostin antibody to detect bone formation via microscopy. Rabbit anti-periostin/HRP (bs-4994R-HRP) and HRP-goat anti-rabbit IgG (074-15-06) were purchased from Bioss (China) and KPL (U.S.), respectively. 2.4. Statistical analysis All test results are expressed in groups in terms of ALP, OT, VEGF, and collagen I levels, and relevant indicators about bone volumes of the femoral head defect area were compared using Student's t-test after the homogeneity of variance was confirmed. Statistical
Fig. 2. Establishment of ONFH in New Zealand white rabbits at 1 month after surgery. ONFH was observed in the experiment group as measured by micro-CT analysis: B-C) sagittal cross-section views, magnetic resonance imaging: D-E) sagittal cross-section views, H&E staining: F-G. A: The subchondral bone of the femoral head was dark red, and the black arrow shows osteonecrosis. B: untreated group. C: treated group, the blue arrow points to the area of ONFH. D: untreated group. E: treated group, the blue arrow points to the area of ONFH. F: untreated group. G: treated group, almost no stained nuclei were observed (black arrow).
analyses were performed by SPSS 21.0 software. P<0.05 was defined as a statistically significant difference. All data are represented as means ± SD (standard deviation). 3. Results 3.1. The establishment of the animal model for ONFH The subchondral bone of the femoral head in the experimental group presented the establishment of the ONFH animal model because of the dark red area on the surface of the femoral head, while the untreated group appeared normal (Fig. 2A). To confirm the model, micro-CT and MRI analysis were employed to detect the specific site. Compared to the untreated group (Fig. 2B), in the treated group, the trabecula of the subchondral bone was disordered and sparse and the outermost bone and the osteoepiphysis
Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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were thin (Fig. 2C). In addition, the femoral head of the treated group (Fig. 2E) showed irregular high-and low-mixed signal areas, which indicates the occurrence of ONFH, and bone signals in the untreated group were uniform (Fig. 2D). The results of H&E staining (40 times magnification) showed that the bone sag became empty, and almost no stained nuclei were observed (Fig. 2G). Conversely, the stroma and nuclei were evenly stained, and there was no nucleus pyknosis in the untreated group (Fig. 2F). 3.2. Self-healing hydrogel supports MC3T3-E1 growth The number and viability of MC3T3-E1 cells was determined using CCK-8 reagent on days 1, 3, 5, and 7. The optical density value positively correlated with the cell number. Therefore, the number of cells gradually increased in the experimental group over time and was close to that of the blank control group (Fig. 3A). The result clearly illustrated that this composite possesses good biocompatibility. 3.3. In vitro osteogenic potential of self-healing hydrogel Other than the biosafety of the gel, its biological effectiveness is of great importance. A series of osteogenic differentiation indicators were assessed. As shown in Fig. 3B, the expression levels of every indicator in the material group were significantly higher than those in the blank group, with a statistically significant difference. The results of the ALP and Alizarin Red staining were promising. The number and density of calcium nodules in the material group was clearly increased compared to the control group (Fig. 3CeF). The area of stained ALP in the experimental group was significantly greater than that in the control group. All the experimental results above indicate that the hybrid hydrogel can support the growth of MC3T3-E1 cells and also promote the osteogenic differentiation of MC3T3-E1 cells in vitro. 3.4. Bone regeneration facilitated by implantation of injectable hydrogel 3.4.1. Micro-CT evaluation Fig. 4 shows that the amount of bone regeneration at 1 and 2 months after injection of HA-BP/CaP in the experimental group was significantly greater than that in the control group. The amount of regenerated bone at 2 months after surgery was significantly greater than that at 1 month post-surgery (Fig. 4AeE).
Fig. 4. A-D: 3D reconstruction views, A corresponds to 1 month after surgery without hydrogel placement; B to 1 month after hydrogel implantation; C corresponds to 2 months after surgery without hydrogel placement; D to 2 months after hydrogel implantation. E: Quantification of the newly formed bone volume per osteonecrosis area. H&E after hydrogel implantation for 1 month (G) and 2 months (I), without hydrogel for 1 month (F) and 2 months (H). Black arrows: collagen, white arrows: osteoblast cells. The scale bar is 100 after hydrogel implantation for 1 month (was perioK) and 2 (M) months, without hydrogel for 1 month (J) and 2 months (L). More swirling bone units were easily found in the implanted group (K, M). White arrows: collagen I. The scale bar is 50 he implanted group r is 100 after hydrogel implantation for 1 moO) and 2 (Q) months, respectively. Black arrows: periostin, red arrows: collagen.
3.4.2. Histological and immunohistochemical analysis It was observed that at 1 and 2 months, the amount of regenerated collagen and the number of osteoblasts in the HA-BP/CaP group were higher than in the control group after decalcification and H&E staining. Collagen content in the material group at 2 months was significantly higher than that at 1 month and was the same as that in the control group (Fig. 4FeI). After staining with Sirius Red and observation by polarized light microscopy, a large quantity of newly regenerated type I collagen was observed (type I collagen fibers are closely arranged, showing a strong birefringence and yellow, orange, and red coarse fibers), which further verifies the results of the H&E staining (Fig. 4J-M). Through immunohistochemical staining, it was observed that the hyperchromatic linear region around the bone trabeculae was periostin, and the expression of periostin at 1 and 2 months in the experimental group was significantly higher than that in the control group. The expression level in the experimental group at 2 months was higher than that at 1 month and was the same as that in the control group (Fig. 4NeQ). 4. Discussion
Fig. 3. The biocompatibility and role in promoting osteogenesis differentiation of HABP/CaP. A: CCK-8 results for biocompatibility. B: The expression of ALP, OT, collagen I, and VEGF in MC3T3-E1 cells on the hybrid hydrogel on day 14. C/D: white arrow: calcium nodules, ODM: osteogenic differentiation medium. E/F: white arrow: the sites of alkaline phosphatase. *p < 0.05, **p < 0.01, ***p < 0.001.
A non-covalent crosslinked hybrid hydrogel with self-healing, shear-thinning, and strong adhesion properties was successfully synthesized [11]. Additionally, this material could boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. Once injected into the site of ONFH in a clinically relevant animal model, it could intensely accelerate bone regeneration. Using an ONFH
Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097
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animal model, we confirmed that this biomaterial can quickly promote bone regeneration in the osteonecrotic area other than healthy bone defect. This finding is promising because it could be an ideal strategy for certain ONFH patients who need to undergo a total hip arthroplasty in the next few years. In recent years, more self-healing gels have been synthesized based on the biocompatibility of HA. However, there are relatively few studies on the use of gels [12]. HA-based composites formed by irreversible covalent bonds exist but unfortunately are not injectable or self-healing [13] and are rarely applied in the field of bone regeneration [14]. Although the focus of few researchers has been on bone or cartilage regeneration [15], one of the critical drawbacks in these studies is that the materials were applied to healthy bone or cartilage defects. The animal models are inconsistent with the pathophysiological mechanisms observed in patients, especially in ONFH. In the present study, the New Zealand rabbit model of ONFH was produced. Clinical core decompression surgery and bone grafting were simulated in the animal model, and bone grafts were replaced with the HA-BP/CaP. This material has significant advantages in clinical use: simpler operation, small wound on the skin and the surrounding soft tissues, shorter operation time, and matching of the shape of the injected hydrogel to that of the bone defect area [16e18]. The conditions for bone regeneration are determined by the interaction between the cells in the bone defect area and the surface of the implant materials. Once the hybrid hydrogel is implanted into the animal, the immune system will recognize the antigenic determinant presented on the HA-based composite, and then, the immune system will probably reject the implant. Hence, its use will not facilitate bone regeneration on account of immunological rejection. Accordingly, one of the keys to success in this approach is reducing the immunological rejection and increasing the biocompatibility of the biomaterials [19]. The results of the CCK-8 assay proved the excellent compatibility of HA-BP/CaP. In addition to the biosafety of the gel, its biological effectiveness is of great importance. A series of osteogenic differentiation indicators were assessed. ALP, OT, collagen I, and VEGF contents were estimated by Q-PCR. In addition, ALP and Alizarin Red staining were used to evaluate the calcium nodules and the amount of ALP. All the experimental results showed that the hybrid hydrogel can support the growth of MC3T3-E1 cells and also promote the osteogenic differentiation of MC3T3-E1 cells in vitro. HA-BP/CaP contains a large number of proteins, with a threedimensional structure that can mimic the extracellular matrix [20]. Calcium phosphonate, a natural component of bone, possesses osteoinductivity and osteoconductivity [21]. BPs are widely used clinically in the treatment of osteoporosis and are also incorporated as bioactive molecules into a variety of bio-scaffolds to promote bone regeneration [22]. BPs have the property of targeted binding of bone minerals. Therefore, we predicted that the material can enhance tissue regeneration. Based on the animal model, New Zealand white rabbits were treated according to the procedure that is applied in clinical practice to treat patients with ONFH [23]. Bone regeneration was assessed by micro-CT and histology. Compared to our reported biomaterial (Am-HA-BP.CaP@mSF) [24], HA-BP/CaP exhibits excellent biological properties and its osteogenic differentiation and bone regeneration promoting abilities are similar to those of Am-HA-BPnatural component of bone, possesses osteoinductivity and osteoconductivitye HA-BP/CaP was applied to an osteonecrotic bone area. Additionally, more tests were performed to confirm new bone formation by CT, collagen by H&E staining, collagen I by Sirius red staining, and periostin by immunohistochemistry. The most abundant organic component in bone is collagen I [25]. The periostin in bone tissue is mainly expressed in
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the periosteum. It controls many stages of intramembranous osteogenesis by facilitating osteoblast cell adhesion, proliferation, and differentiation [26,27]. To determine the impact of HA-BP/CaP on osteogenic differentiation in vitro, Q-PCR was used and extracellular matrix mineralization indicators, such as calcium nodules and ALP, were assessed. Interestingly, all results illustrated the excellent biological effectiveness of ONFH in promoting osteogenic differentiation in vitro and bone regeneration in vivo. 5. Conclusion A non-covalent crosslinked hybrid hydrogel with self-healing, shear-thinning, and strong adhesion properties was successfully synthesized. Additionally, this material can boost proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. Once injected into the site of ONFH in the novel, clinically relevant animal model, it can intensely accelerate bone regeneration and is a promising tissue engineering strategy. In the future, other bioactive molecules may be loaded onto the biomaterial to further improve the performance of this material, such as VEGF and bone morphogenetic protein-2. Conflicts of interest The authors declare that they have no competing interests. Acknowledgments We acknowledge financial support from 1) Beijing natural science foundation youth project NO.7184235; 2) China postdoctoral foundation NO.62 general program. References [1] K.J. Guo, et al., The influence of age, gender and treatment with steroids on the incidence of osteonecrosis of the femoral head during the management of severe acute respiratory syndrome: a retrospective study, Bone Joint Lett. J 96b (2) (2014) 259e262. [2] A. Wang, M. Ren, J. Wang, The Pathogenesis of Steroid-induced Osteonecrosis of the Femoral Head: a Systematic Review of the Literature, Gene, 2018. [3] J.R. Lieberman, et al., Osteonecrosis of the hip: management in the 21st century, Instr. Course Lect. 52 (2003) 337e355. [4] K. Ikeuchi, et al., Epidemiology of nontraumatic osteonecrosis of the femoral head in Japan, Mod. Rheumatol. 25 (2) (2015) 278e281. [5] P. Luo, et al., The role of autophagy in steroid necrosis of the femoral head: a comprehensive research review, Int. Orthop. 42 (7) (2018) 1747e1753. [6] J.S. Kang, et al., The natural history of asymptomatic osteonecrosis of the femoral head, Int. Orthop. 37 (3) (2013) 379e384. [7] D. van der Jagt, et al., Osteonecrosis of the femoral head: evaluation and treatment, J. Am. Acad. Orthop. Surg. 23 (2) (2015) 69e70. [8] Y.A. Fillingham, B.A. Lenart, S. Gitelis, Function after injection of benign bone lesions with a bioceramic, Clin. Orthop. Relat. Res. 470 (7) (2012) 2014e2020. [9] C.G. Finkemeier, Bone-grafting and bone-graft substitutes, J Bone Joint Surg Am 84-a (3) (2002) 454e464. [10] M. Putzier, et al., Allogenic versus autologous cancellous bone in lumbar segmental spondylodesis: a randomized prospective study, Eur. Spine J. 18 (5) (2009) 687e695. [11] M.R. Nejadnik, et al., Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles, Biomaterials 35 (25) (2014) 6918e6929. [12] Q. Wang, et al., Injectable PLGA based colloidal gels for zero-order dexamethasone release in cranial defects, Biomaterials 31 (18) (2010) 4980e4986. [13] B. Rybtchinski, Adaptive supramolecular nanomaterials based on strong noncovalent interactions, ACS Nano 5 (9) (2011) 6791e6818. [14] Q.G. Wang, et al., Molecular profiling of single cells in response to mechanical force: comparison of chondrocytes, chondrons and encapsulated chondrocytes, Biomaterials 31 (7) (2010) 1619e1625. [15] Hou, S., et al., Rapid self-integrating, injectable hydrogel for tissue complex regeneration. Adv Healthc Mater, 2015. 4(10): p. 1491-1495, 1423. [16] P. Ni, et al., Injectable thermosensitive PEG-PCL-PEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects, Biomaterials 35 (1) (2014) 236e248. [17] K. Bergman, et al., Injectable cell-free template for bone-tissue formation, J. Biomed. Mater. Res. 91 (4) (2009) 1111e1118.
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Please cite this article as: Y. Wang et al., Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.097