Temperature-sensitive, 1- bromoheptafluorooctane-containing hydrogels in repairing bone defect in rabbits

1 Introduction

Bone defects are among the common consequences of bone trauma. Recently, the application of biomaterials for bone regeneration has become a hotspot in this field (Kang et al., 2014; Stevens, 2008). Bone defects pose significant challenges in orthopedics, affecting patients well-being and mobility. These defects result from various factors, including traumatic injuries, bone diseases, and congenital abnormalities, leading to pain and functional limitations that diminish the quality of life (Ricci et al., 2007). However, the complex nature of bone tissue and the intricacies of bone regeneration necessitate innovative approaches and advanced biomaterials for successful bone defect repair (Dimitriou et al., 2005)- (Khan et al., 2000).

Bone grafts serve as essential scaffolds or matrices that facilitate the formation of new bone tissue, expediting the healing process and restoring the structural integrity of the affected area (Patzakis et al., (1976–2007) 1983). The need for such advanced graft materials arises due to the significant limitations and challenges associated with bone defects, which may originate from traumatic injuries, bone diseases, or congenital abnormalities. Carefully selecting an appropriate bone graft material is paramount in achieving successful outcomes in bone grafting procedures (Einhorn, 1995). The chosen graft material must possess crucial characteristics, such as biocompatibility, osteoconductivity, and osteoinductivity. Biocompatibility ensures seamless integration within the recipient's body without causing adverse reactions, while osteoconductivity promotes cell migration and new bone formation. Osteoinductivity stimulates mesenchymal stem cells differentiation into bone-forming cells, facilitating bone regeneration (Einhorn, 1995).

Numerous bone graft materials are available, including autografts, allografts, xenografts, and synthetic grafts. Autografts from the patient's body are considered the gold standard due to their exceptional biocompatibility and osteogenic properties (Rodríguez-Merchán, 2022). Allografts and xenografts offer viable alternatives when autografts are not feasible, provided they undergo rigorous processing to minimize immune responses and disease transmission. Synthetic graft materials, like ceramics, polymers, and composites, offer versatility and customization options but may require additional biological factors to enhance bone regeneration. In conclusion, the judicious selection of the appropriate bone graft material is pivotal in optimizing bone grafting procedures and improving patient outcomes (Zhao et al., 2021).

Temperature-sensitive hydrogels have emerged as highly promising biomaterials with significant potential for bone graft applications as they undergo a sol–gel phase transition in response to temperature changes, solidifying at physiological temperatures (Yi et al., 2023). This unique property enables their minimally invasive injection or application in a liquid state, followed by in-situ solidification, conforming precisely to the bone defect site. The tunable nature of temperature-sensitive hydrogels allows customization of their properties to match specific clinical needs. Combining them with bioactive agents or stem cells further augments their regenerative potential. Although ensuring biocompatibility and evaluating the host response is crucial before clinical translation, ongoing research suggests that temperature-sensitive hydrogels hold transformative prospects for bone grafting, fostering improved patient outcomes and advancing regenerative therapies in orthopedics (Feng et al., 2019).

The utilization of temperature-sensitive hydrogels in bone graft materials confers several advantages. Their liquid form enables minimally invasive delivery, ensuring precise placement within complex defect sites. Upon reaching body temperature, the hydrogel solidifies, securing its position and preventing leakage. Their excellent biocompatibility and capability to encapsulate bioactive molecules, growth factors, or cells make them particularly appealing for promoting bone regeneration. This multifaceted approach, leveraging the sol–gel transition, showcases the immense potential of temperature-sensitive hydrogels in advancing bone grafting strategies and enhancing regenerative therapies in orthopedics (Lavanya et al., 2020; Zeng et al., 2022).

Combining temperature-sensitive hydrogels with perfluorocarbons (PFCs) for bone defect repair is a relatively unexplored area in the literature. This study assesses their potential synergistic effects in bone regeneration and their impact on early-stage hypoxia. The investigation will evaluate the effectiveness of temperature-sensitive hydrogels incorporating PFCs in repairing bone defects and enhancing healing. Rigorous experimental methodologies, including in vitro and in vivo assessments, will characterize the materials and elucidate their mechanisms (Sun et al., 2020). The study seeks to provide valuable insights into their clinical application and potential therapeutic advancements. By understanding the interaction between the two components, novel graft materials may be developed for more effective bone regeneration. Ultimately, these findings can potentially revolutionize bone defect repair strategies and improve clinical practices in orthopedic surgery and regenerative medicine (Wang et al., 2023).

Bone regeneration is an oxygen-consuming process, and the occurrence of early hypoxia in the defect area hinders the processes of bone regeneration and healing (Liu et al., 2015; Stiers et al., 2016). To overcome this problem, researchers have studied the incorporation of perfluorocarbons (PFCs) into various bone graft materials (Tamimi et al., 2013; Wang et al., 2013; Ma et al., 2013; Zhu et al., 2014). Temperature-sensitive chitosan hydrogels are novel bone graft materials, which can be solidified at 37℃. Despite their poor mechanical properties, these hydrogels can be used in constructing the ideal composite bone graft materials due to their convenient configuration (easy shaping of the hydrogel in different conditions), easy operation (easy use in different situations), and good histocompatibility (Saravanan et al., 2018; Saravanan et al., 2019).

However, the combination of temperature-sensitive chitosan hydrogels and PFCs in repairing bone defects has not been studied yet. This study investigated the potential of 1-bromoheptafluorooctane-containing, temperature-sensitive chitosan hydrogel in repairing the femoral condyle bone defect in rabbits, and improving the early-stage, post-transplantation hypoxia, as well as the possible mechanism of healing.

2 Materials and methods

3 Results

3.1

3.1 General observations

At four weeks post-operation, there was no sign of necrosis in and around the bone defect area in groups A and B. The bone cavity area was significantly reduced as the cavity was filled with hard tissue with light-brown color. However, the hardness of the newly formed bone tissue was poor. The area of the bone cavity in group C was significantly larger compared to the other two groups, and the cavity was filled with soft tissue with dark red color. At twelve weeks post-operation, the majority of the bone defect areas in groups A and B were filled with newly regenerated bone tissue, and cortical bone was formed on the surface of the defects. In group C, the bone cavity was still present and filled with a milky white-colored soft tissue (Fig. 1).

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Fig. 1

General observation of different treatment groups. A, B, and C represent groups A, B, and C at four weeks post-operation and D, E, and F represent groups A, B, and C at 12 weeks post-operation, respectively.

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3.2

3.2 Analysis of X-ray scans

At Four weeks post-operation, a high-density shadow representing newly formed bone tissue was observed in the defect areas of groups A and B. The gray value of group A was slightly higher compared to group B, while no bone density shadow was observed in group C. At 12 weeks post-operation, a low-intensity bone density shadow was observed in the defect area of group C, however the round outline of the cavity was still visible, and the sclerotic zone was formed at the edges of the cavity. On the contrary, the high-density shadow of newly regenerated bone tissue was observed in the cavities of groups A and B, which had no significant difference with the surrounding cancellous bone (Fig. 2).

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Fig. 2

X-ray imaging of each group. A, B, and C represent groups A, B, and C, at four weeks post-operation and D, E, and F represent groups A, B, and C at 12 weeks post-operation, respectively.

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3.3

3.3 Micro-CT

3D reconstruction of Micro-CT, sagittal sectional images, and newly formed trabeculae in each group showed that at four weeks post-operation, the area of the bone cavity in group A was significantly smaller compared to the other two groups, indicating the growth of new trabeculae in the defect cavity. The volume and quality of the newly formed bone tissue were superior to the other two groups. However, the area of the bone cavity in group C was still noticeable, and no bone regeneration was observed at the center of the cavity. At 12 weeks post-operation, the bone cavity disappeared in groups A and B. There was no significant difference between the number and shape of trabeculae in the cavity and surrounding cancellous bone. However, the maturity of the new trabeculae and the hardening degree of the cortical bone in group A were superior to other groups. In group C, the bone cavity still existed, and only a small amount of bone was regenerated around the bone cavity (Fig. 3).

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Fig. 3

Micro-CT imaging results. A, B, and C represent groups A, B, and C, at four weeks post-operation and D, E, and F represent groups A, B, and C at 12 weeks post-operation, respectively.

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Quantitative analysis showed that TMD, BVF, Tb.Th and Tb.N in group A were significantly higher compared to the other groups at four and 12 weeks post-operation. However, there was no significant difference in SMI values between groups A, B, and C at four weeks post-operation. The SMI values of groups A, B, and C showed significant differences at 12 weeks post-operation in the following order: group A < group B < group C (Table 1).

Table 1 Micro-CT quantitative data analysis of each group at each time point post-operation (χ s).

		Group A	Group B	Group C
Bone mineral density (TMD) mg/cm3	4 w	496.120 ± 4.960	479.100 ± 5.890b(bp = 0.00002010)	468.480 ± 3.650ab(ap = 0.00000005；bp = 0.00171183)
	12 w	579.800 ± 6.570	562.730 ± 9.230b(bp = 0.00063886)	513.670 ± 3.710ab(ap = 2.9263E-09；bp = 4.4451E-11)
Bone volume fraction (BVF)	4 w	15.730 ± 1.050	12.770 ± 1.010b(bp = 0.00005679)	10.200 ± 0.680ab(ap = 0.00022310；bp = 3.1205E-08)
	12 w	51.010 ± 1.960	45.100 ± 3.600b(bp = 0.00296843)	23.160 ± 2.870ab(ap = 1.2281E-09；bp = 4.2689E-11)
Bone trabecular thickness (Tb.Th) mm	4 w	0.125 ± 0.004	0.106 ± 0.006b(bp = 7.4045E-06)	0.090 ± 0.005ab(ap = 0.00004863；bp = 3.0900E-09)
	12w	0.251 ± 0.006	0.209 ± 0.017b(bp = 0.00002444)	0.142 ± 0.010ab(ap = 1.1299E-07；bp = 1.3488E-10)
Trabecular bone number (Tb.N) 1/mm	4 w	0.900 ± 0.020	0.700 ± 0.030b(bp = 2.1962E-08)	0.520 ± 0.050ab(ap = 1.2693E-07；bp = 3.2721E-12)
	12 w	3.530 ± 0.050	3.100 ± 0.030b(bp = 2.3637E-11)	1.780 ± 0.050ab(ap = 1.9162E-18；bp = 2.7041E-20)
Structural model index (SMI)	4 w	1.900 ± 0.080	1.950 ± 0.100d(dp = 0.43352368)	1.990 ± 0.110 cd(cp = 0.54076756；dp = 0.17301768)
	12 w	0.070 ± 0.030	0.510 ± 0.040b(bp = 1.7415E-11)	1.280 ± 0.050ab(ap = 4.9034E-15；bp = 5.9441E-18)

Note: At the same sampling time point, compared with group B, ^ap < 0.05; Compared with group A,^b p < 0.05; Compared with group B,^c p > 0.05; Compared with group A,^d p > 0.05.

3.4

3.4 Masson’s trichrome staining

At four weeks post-operation, the bone cavity in group C was filled with a large number of inflammatory cells and a small amount of fibrous tissue. However, newly regenerated bone tissue as well as scattered chondrogenic centers could be observed in the bone cavities of groups A and B, while a large number of osteoblasts and collagen fibers were present in the osteogenic centers. At 12 weeks post-operation, the bone cavity still existed in group C, the edge of the cavity was sclerotic, the bone trabecula was thick, and the enlarged sclerotic zone showed red and blue lamellar bone structure. The bone defect areas of groups A and B were filled with newly generated bone tissue, while the trabecula of group A was more mature, with a large number of mature red-stained matrices.(Fig. 4).

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Fig. 4

Masson’s trichrome staining results of each group. A, B, and C represent groups A, B, and C, at four weeks post-operation and D, E, and F represent groups A, B, and C at 12 weeks post-operation, respectively.

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3.5

3.5 Immunohistochemical staining and micro-vessel count

At four and 12 weeks post-operation, CD31 was expressed in the soft tissues of the bone marrow cavity of the regenerated trabeculae in all groups, however, the number of micro-vessels in group A was significantly higher compared to the other two groups (Fig. 5 and Table 2).

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Fig. 5

Immunohistochemical staining. A, B, and C represent groups A, B, and C, at four weeks post-operation and D, E, and F represent groups A, B, and C at 12 weeks post-operation, respectively.

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Table 2 Micro-vascular number in each group at each time point post-operation (the number of micro-vessels/200 times visual field, χ s).

	4 weeks	12 weeks
Group A	5.90 ± 0.22	15.62 ± 0.29
Group B	3.08 ± 0.26b(bp = 1.2185E-11)	13.20 ± 0.34b(bp = 2.0892E-9)
Group C	0.65 ± 0.30ab(ap = 7.6483E-11；bp = 1.1796E-15)	5.57 ± 0.36ab(ap = 1.1844E-16；bp = 1.9671E-18)

Note: Compared with group B,^ap < 0.05 at each time point post-operation; Compared with group a,^bp < 0.05.

4 Discussion

Bone regeneration is an energy-consuming process requiring oxygen. Due to high oxygen consumption during bone regeneration, the oxygen supply in the bone defect area deteriorates rapidly following bone injury. This high rate of oxygen consumption cannot be compensated by oxygen penetration from the blood vessels of the surrounding tissues, which leads to the sudden drop of local oxygen partial pressure following the occurrence of bone injury, making bone regeneration extremely difficult (Drager et al., 2015; Park et al., 2019). The authors believe (Pacheco et al., 2017) that following the occurrence of bone defect, restoring the oxygen level in the bone defect area, can effectively promote bone regeneration. In clinical practice, bone flap transplantation with blood supply has solved the oxygen problem with good therapeutic outcomes. However, this approach is not widely used due to technical difficulties. Following the development of bone tissue engineering, researchers have used perfluorocarbon, as an oxygenating agent, to provide sufficient oxygen supply in the bone defect area, and to promote bone regeneration (Allen et al., 2016; Hwang et al., 2019).

We have previously shown that the temperature-sensitive chitosan hydrogel containing 1-bromoheptafluorooctane, developed in our lab, has suitable physical and chemical properties, oxygen evolution performance, histocompatibility, and degradability (Lin et al., 2013). The hydrogel degrades completely in vivo within six weeks, in which the oxygen-enriched agent remained stable and not degraded, and was excreted through the lungs. Chitosan is metabolized into acetylglucosamine in vivo, which is not only safe and non-toxic, but it can directly provide energy for osteogenesis, and promote osteoblast growth (Wang and Stegemann, 2010). The temperature-sensitive chitosan hydrogel has been widely used in bone tissue engineering due to its convince for injection during surgery and the ability to fill the defect gap (Zhao et al., 2018). Therefore, it is of certain interest to investigate its potential in improving bone repair in animal models.

In this experiment, the oxygen-enriched temperature-sensitive chitosan hydrogel containing oxygen-increasing agent 1-bromoheptafluorooctane was used to repair the femoral condyle bone defect of rabbits and showed a significant bone repair effect. At 12 weeks post-operation, there was no significant difference in X-ray density between the bone defect area and the surrounding cancellous bone in group A. In Micro-CT examination, the TMD in A group were 1.04- and 1.03-fold higher compared with those in B group at four and 12 weeks post-operation, respectively (p < 0.05); The BVF in A group were 1.23– and 1.13-fold higher compared with those in B group at four and 12 weeks post-operation, respectively (p < 0.05); The Tb.Th in A group were 1.18- and 1.2-fold higher compared with those in B group at four and 12 weeks post-operation, respectively (p < 0.05); The Tb.N in A group were 1.29- and 1.14-fold higher compared with those in B group at four and 12 weeks post-operation, respectively (p < 0.05). The bone histomorphological staining also showed that the bone defect area was filled with more new bone, compared to the other two groups, indicating a higher mineralization degree. When osteoporosis occurs, the bone trabeculae transform from plate-like to rod-shaped, and the SMI value increases. In this experiment, we also found that the SMI value of the new bone in group A, defined the degree of bone trabecular plate and rod like shapes, was significantly lower compared to the other two groups at 12 weeks post-operation, indicating that the trabecula of the new bone was more mature. In our experiment, the trabecula of group A was more mature, with a large number of mature red-stained matrices. The microstructure of the trabecula of group B was more naive, and many new light-blue matrices. However the bone cavity still existed in group C, the edge of the cavity was sclerotic, the bone trabecula was thick, and the enlarged sclerotic zone showed red and blue lamellar bone structure (Fig. 4). It means that the bone trabeculars in perflubron group was more maturer. All these findings indicate that the perflubron enhanced bone formation and maturation in vivo. Researchers have also prepared fibrin hydrogels containing fluorocarbon (as oxigenating agent) seeded with bone marrow stromal cells, and observed improved survival and activity of these cells, as well as a substantial improvement in ectopic osteogenesis and bone repair (Kimelman-Bleich et al., 2009; Benjamin et al., 2013). Consistent with our findings, these results confirm that introducing an oxygen-increasing agent into bone tissue engineering materials can fix early hypoxia following bone graft transplantation. This approach also improved the functionality of seeded cells and yielded superior bone healing effect. In addition, we found that the number of micro-vessels in A group were 1.92- and 1.18-fold higher compared with those in B group at four and 12 weeks post-operation, respectively (p < 0.05); the number of micro-vessels in A group were 9.08- and 2.8-fold higher compared with those in C group at four and 12 weeks post-operation, respectively (p < 0.05).. This might be the result of improved oxygen supply in the early stage of graft transplantation, supporting the survival of new functional cells migrating into the defect area and the subsequent release of factors such as VEGF, which could accelerate the vascularization of bone graft materials (Hu and Olsen, 2016). Further investigations are needed to elucidate completely the mechanism through which 1-bromoheptafluorooctane exerts its positive effect on new functional cell viability and the vascularization of bone graft materials.

In this research, the temperature-sensitive chitosan hydrogel containing 1-bromoheptafluorooctane was used as a scaffold and an oxygen carrier in the process of bone repair, which not only promoted the growth of osteoblasts and new blood vessel formation but also provided sufficient oxygen supply for early osteogenesis. However, due to the poor mechanical strength of the hydrogel, it is not feasible to use it as a bone graft substitute alone to repair bone defects. In addition, the oxygen-enriched water gel has a limited time to release oxygen, which can't effectively provide the required oxygen supply for complete osteogenesis in the later stages of the healing process. Therefore, combining the oxygen-enriched hydrogel with other bone tissue engineering materials with superior mechanical properties is encouraged.

However, there are some limitations in this study. We did not analyze mechanical properties of the temperature-sensitive hydrogel containing 1-bromoheptafluorooctane. The detail characterization of the scaffold material analysis at each time point would provide more information on bone formation in our model. Further studies are needed to investigate the effect of perflubron-induced oxygen elevation on the molecular events of new functional cells migrating into the defect area during bone formation in vivo, which will aid us in understanding the effects of perflubron on bone regeneration. On the other hand, the roles of perflubron in bone graft vascularization are still unclear. The vascularization of bone graft is also a crucial process for bone regerneration, which could supply oxygen and nutrient within the surrounding medium at later stage after implantation. There will be a beneficial effect of vascularized bone scaffold supplemented with “PFCs-MSCs combinations” on bone regeneration in animal models of bone defect. Above combined usage is expect to provide temporary sufficient oxygen for MSCs in the early stage and supply more stable oxygen and nutrients by vascular network in the latter stage, which might achieve a better bone regeneration. In conclude, more investigations are needed before application of the oxygenated thermoresponsive hydrogel in clinical settings.

Perflubron, as the main component of Oxygent(TM), has been known as blood substitutes in the field of blood tissue engineering. On account of its high solubility of oxygen, investigators have shown gaining interest in exploring its use in tissue engineered devices. Thus far, it has not been investigated that combining perflubron with thermoresponsive hydrogels for enhancing bone formation in vivo. In conclusion, the temperature-sensitive chitosan hydrogel containing 1-bromoheptafluorooctane can effectively promote the repair of rabbit femoral condyle bone defects and can be considered a promising bone tissue engineering material.