Radiological Evaluation Of Acellular Fish Swim Bladder and Autologous Bone Marrow Clot on Induced Radial Bone Defects in Rabbits

The repairing of segmental bone defects was stilled an important challenging for orthopedic surgeons and usually the segmental bone defects were resulted from trauma, infection, bone tumors resection and congenital malformation (Pereira et al., 2020; Huang et al., 2020). The preferred treatment option for the healing of bone defects is bone grafting which it was useful in the management of delayed union, nonunion and arthrodesis (Jonitz et al., 2011). Clinically autograft, allograft and xenograft had been utilized as graft substitutes (Mollon et al., 2013). Autograft is the gold standard for bone grafting (Wubneh et al., 2018). A variety of treatment options were developed for segmental bone defects in rabbits’ radius bone such as Masquelet technique, which it was involved the first creating of a cement spacer to induce a membrane, then it was filled with bone graft material in a second stage; alternatively, bone substitutes like avian eggshell hydroxyapatite powder, α-calcium sulfate hemihydrate (α-CSH), and porous tantalum scaffolds were showed a promising in treatment rabbits’ bone defects (Wang et al., 2017; Meng et al., 2019; Atiyah et al., 2020). Furthermore, in rabbits with critical-sized radial lesions and decellularized bone matrix (DBM) scaffolds filling with platelet-rich plasma (PRP) were also utilized to enhance bone the integration and regeneration (Leng et al., 2020). Critical advances were conducted in the bone tissue engineering field and they were fully determined during supplanting the utilization of autogenous bone grafts to keep away from the adverse consequences lead toimproving the regeneration of bone (Peric et al., 2015).

Extracellular matrix (ECM) derived biomaterials had been clinically utilized in the surgery lead to repairing different tissues and organs (Al-Falahi, 2009; Al-Falahi et al., 2016; Al-Bayati et al., 2016; Al-Falahi et al., 2017; Al-Ebadi and Al-Bayati, 2019; Gumaa and AL-Bayati, 2021; Mohammed and Salih, 2022; Mohammad and Al-Ebadi, 2023). Different types of whole ECM were utilized as biologic scaffolds to encourage the remodeling of organs and tissues; these ECM scaffolds were commonly gathered from the small intestine, urinary bladder, skin, pancreas and liver (Badylak, 2007).

Other studies were demonstrated that scaffolds mimicking the mechanical properties of natural bone can be enhanced the integration and stability of the newly formed bone (Wang et al., 2017). The extracellular matrix was essential in the repairing of segmental bone defects in rabbits, facilitating cellular activities and providing mechanical supporting and stability to the defect site, which was a vital for load-bearing during the healing process lead to an enhancement of the biological processes such as healing. Osteoblasts and mesenchymal stem cells (MSCs) were two examples of the bone cells that may be proliferated more easily when they are supported by the extracellular matrix (ECM). While the ECM was stimulated the growth factors and signaling molecules lead to control on cellular function, this was necessary to start the mending process in bone abnormalities. These elements were affected on MSCs’ ability to differentiate into osteoblasts, which they were essential for the production of new bone. An improvement of repairing bone can be resulted from ECM components by increasing osteogenic capacity. In addition the ECM components were also contributed to bone remodeling. The ECM altered as new bone forms, making it easier to shift from a temporary scaffold to a more permanent one. Restoring the mechanical integrity of the bone depended on this remodeling. Several biocompatible scaffolds such as collagen and peptide-modified scaffolds were utilized to improve the bone healing in experimental animals. Scaffolds were intended to an enhancement the overall healing of segmental bone defects. (Lin and colleagues, 2020).

Fish swim bladder (FSB) collagen was considered an essential constituents of the ECM, it had the advantages of low immunity, significant biocompatibility and biodegradability, easily available, low cost material and easily decellularized, it was mainly composed from collagen I with low cellularity compared to other tissues with extremely strong mechanical strength because of its inflation function, which it was a crucial during tissue regeneration (Mredha et al., 2017). Collagen membranes from fish swim bladders were used as a strong and acid-resistant suture for pH-regulated stomach perforation and tendon rupture and research on biomaterials for tissue engineering by using appropriate scaffold from FSB as an application of vascular graft (Bai et al., 2022; Luan et al., 2022). Cell-based therapies were one way to enhance the healing process of injured tissues. The three primary approaches were developed and utilized for tissues regeneration, these an important approaches were included bone marrow aspirate (BMA), bone marrow concentrate and cultured mesenchymal stem cells (MSCs) (Veronesi et al., 2013). Bone marrow aspirating was founded to be a great source of endogenous reparative cells and growth factors for the successful repairing of bone, cartilage and soft tissues (Gangji, et al., 2011; Veronesi et al., 2017; Lim et al., 2019; Thanoon et al., 2019). Bone Marrow Aspiration BMA was a direct aspiration of autologous bone marrow from iliac crest and long bones such as humerus or femur. BMA tended to coagulate due to the presence of megakaryocytes, platelets and coagulation factors. There was a strong reasoning for the use of BMA clot for strategies of tissue regeneration (Salamanna et al., 2018).

Due to the lack of studies on using extracellular matrix from natural sources as scaffolds for bridging segmental bone defects in Iraq; this study focused on converting waste fish swim bladder into valuable scaffold by combining waste fish swim bladder with autologous bone marrow aspirating clot for reconstructive of segmental bone defect.

All procedures in present study were provided ethical approval, from the local committee of animal care and use at the College of Veterinary Medicine - University of Baghdad (No: P.G/1222 at June-26th 2024).

Fifteen healthy adult rabbits weighing 1.5–1.7 kg and aged 9–12 months were used in the current study. They were housed in special cages (60 × 50× 50) centimeters metal crates in the College of Veterinary Medicine. Throughout the trial, these animals were maintained at ordinary room temperature, which it was 22 ± 3°C for 15 days to acclimatization before surgery, they were fed green grass and commercial pellets and all animals were given 0.2 mg/kg. B.W. (S/C) Ivermectin (AVICO, Jordan) (Sivajothi et al., 2014).

The fifteen experimental animals were divided randomly into three groups (n=5), the induction of segmental defects (5 mm) were done in the mid shaft of the radius bone in each animal by using a low-speed electrical saw and continuous irrigation with a 0.9% sterile saline solution, the first group was induced defects and left without any treatment (control group ). The second group was induced defects and treated with fish swim bladder treated group (FSB group). While the third group was induced defects and treated with fish swim bladder with autologous bone marrow clot (FSB-BMC group). The segmental defects in all groups were evaluated radiographically immediately after operation, then every two weeks till 12 weeks post-operation on a medio-lateral view with X-Ray machine (ENIE Radiologie®) at 45 kv and 5 mAs, to observe the bridging of the segmental bone defects and the development of new bone formation as in (Figure 1).

Fresh swim bladders of carp fish were obtained from the local market and transported to the lab in a container with phosphate buffer solution (PBS). After removing the outer fat and blood capillaries. The swim bladders were rinsed and washed with PBS at 37 °C for 15 minutes. Next, the FSB scaffolds were decellularized using chemical decellularization method, which it was lysed the cell membrane and other cellular components with 0.5% sodium dodecyl sulphate (SDS) for 24 hours at room temperature and after that it was gently shaking the container three times (Crapo et al., 2011; Kumar et al., 2015). The decellularized scaffolds were cutting into approximately (1×1.5cm pieces) and terminally sterilized for 2 hours using 0.1% Peracetic acid (PAA) and 4% ethanol solution (Tao et al., 2021). Then the scaffolds were washed for 15 minutes at 37 °C using phosphate buffer solution (PBS) and stored in sterile PBS containing streptomycin (100μg/ml), penicillin (100 IU/ml) and Amphotericin (100μg/ml) and finally the scaffolds were preserved at 4 °C. Samples of FSB after decellularization were obtained and fixed in 10% buffer formalin solution for sectioning and staining using hematoxylin-eosin stain, then the samples were examined histologically after completing removal of cells with wavy dense staining acellular collagen fiber and loose delicate light-stained elastic fibers.

Anesthetic protocol: Acepromazine (1 mg/kg) was injected intramuscularly into the experimental animals to sedate them (Prozil fort, Vietnam). 10% Ketamine (40 mg/kg of ) and 5 mg/kg of 2% Xylazine Hydrochloride (VMD-Belgium) were given I/M and as a general anesthesia after 10 minutes of Acepromazine injection . (Flecknell, 2015).

Bone marrow aspiration: After giving the general anesthesia and undergoing aseptic surgical preparation of the aspiration filed, the animals were placed into a lateral recumbency. A tiny stab skin incision was made for each animal to facilitate entry of the aspiration needle gauge 16 in the mid-way between the head of femur and the greater trochanter. The location of the trochanter fossa was determined by palpating the greater trochanter. The sterile needle of syringe was inserted toward the trochanteric fossa and carefully rotated until reaching to the medullary cavity to obtain 1ml of BM sample (Awid et al., 2014) as in (Figure 2A, B). The aspirating BM was allowed to clot for fifteen minutes before implantation and cut to fit the creating of radial defect in the same animal (Lim et al., 2019) as in (Figure 2C, D).

Following aseptic preparation of the surgical site in the right forelimb from the mid-shift of the humerus to the metacarpal bones, the anesthitized experimental animals were placed in a right lateral recumbency position. A 3–4 cm and skin incision was performed on the right forelimb’s cranio-medial aspect in equivalent distance between the elbow and carpal joint as in (Figure 3A). The pronator teres and flexor carpi radialis muscles were carefully separated with fine blunt scissors to expose the radius bone as in (Figure 3B). Distance (5mm) was marked at the radius mid shaft for osteotomy as in (Figure 3C), the marked segment was removed using electrical saw irrigated and (0.9%) sterile saline solution to create a defect in the radius mid shaft as in (Figure 3D). In the control group the segmental defect was left empty, whereas in the FSB group the defect was wrapped using the previously preparation of acellular FSB and it was sutured as a tube around the radius bone defect as in (Figure 3E), while in the FSB-BMC group the radial defect was wrapped using acellular FSB and filled with autologous bone marrow clot as in (Figure 3F), Subsequently, a simple continuous pattern using 5/0 polydioxanone (PDS) was used to suture the FSB membrane (Figure 3G). The muscles were approximated by simple continuous suture pattern using 3/0 polydioxanone (PDS) and an interrupted suture pattern with 3/0 silk. After that, the skin was closed (Figure 3H). Intramuscular injection of meloxicam at a dose of 1.5 mg/ kg. B.W. was given as analgesia (Ahmed et al., 2017), penicillin-streptomycin antibiotics were administered at doses of 10,000 IU/Kg B.W. and 5 mg/Kg B.W., respectively for 3 days post operation.

Numerous studies were recorded about bone repairing of animal models. Study done by Bigham-Sadegh and Oryan (2015), they mentioned rabbits a good animal to examine the segmental defects and grafts with structural qualities. As well as, previous reports of this model’s suitability had been mentioned rabbits did not required internal or external fixation lead to impact the healing process (Atiyah et al., 2020). Therefore, in current study a radius defect of approximately (0.5) cm was created to evaluate acellular FSB effect as guided bone regeneration to bridging the radius defect associated with autologous BMC in rabbits without needing for any fixation. Numerous research were reported on the application of tissue engineering techniques to repair radial abnormalities in New Zealand white rabbits (Kasten et al., 2008; Bodde et al., 2008; Zhao et al., 2011). After middle radial defects, the intact ulna supported the rabbit’s forelimbs without internal fixation. For bone defect models, the mid-diaphyseal radius can frequently be the exacted site. A radiolucent segmental defect area was appeared at the mid shaft of radius bone in control group immediately post –operation as in (Figure 4A). While, the proximal and distal ends of the radius bone segments were showed a mild periosteal reaction in the second week following the operation as in (Figure 4B). Whereas, the proximal and distal ends of the radius bone segments were appeared an increase in thickness of both ends with callus formation and extending from the proximal end of the radius defect towards the defect center and it was attached with the ulna at ٤th week post –operation as in (Figure 4C). While the callus formation was increased from the proximal end of the radius defect, as well as, it was extended to the center of the defect and fused with ulna at 6th week post –operation as in (Figure 4D). A mild periosteal reaction in the radius defect’s distal end was appeared in the eighth week following the procedure as in (Figure 4E). In addition, the proximal end of callus from the radius defects to its center was continuously grew in along week after the surgery and the periosteal reaction in the distal end of radius defect was increased until 12th week post –operation with obvious radio-ulnar synostosis (Figure 4F). Some researchers hypothesized this reaction could be caused by scratching the surface of the bone and it was perfected for starting a biologic reaction lead to ulnar hypertrophy. Other studies were showed this reaction of the activation in periosteal cambium cells leads to the synthesis of callus matrix (Bodde et al., 2008; Cho et al., 2011). This result may be attributed to a biologic reaction from cells in the surrounding tissues and this explaining of current study was agreed with other studies demonstrated a distinct fusing of the radius and ulna at the sites of defect creation by biological reaction (Kasten et al., 2008). Other study reported by Bodde et al. (2008), who mentioned that the membrane interossea between the two bones and the periosteal were remained from the proximal and distal ends of the defect and this result perhaps due to the charge of the synostosis or fusion between the rabbit’s ulna and radius. According to study mentioned by Torbjörn et al. (2021), they showed the assessments of the osseoinductive and osseoconductive qualities in the investigation were made more difficult by the osseous growth of the ulnar bone and its union with the radius.

In present study A radiolucent segmental defect area of FSB treated group immediately post –operation was seen in (Figure 5G), Furthermore, a little periosteal response at both ends of the radius bone defect was appeared in the second week following surgery as in (Figure 5H). There was an increase in the periosteal reaction and radiopaque foci of newly formed bone was showed in the fourth week following surgery as in (Figure 5I). While, the radius defect’s new bone growth was increased and extended from both ends in the sixth week following the procedure (Figure 5J). As well as, an increase in newly formed bone and extended from both ends of the radius defect towards the central new bone formation foci was appeared in eight weeks after the procedure (Figure 5K). The opacity of new bone formation was increased and extended from both ends of the radius defect towards the central new bone formation foci at 10th week post –operation and 12th week post –operation. Furthermore, The opacity of new bone formation was extended from both ends of the radius defect in a straight direction with partially bridging the radius defect as in (Figure 5L), these results might be related to the heamatoma formation which it was considered as the first an important step for the healing of fractures and it was produced in the acellular FSB tube that it was encircled the defect lead to the production of new bone at the defect’s center and a clear extension of the fracture ends. These outcomes were consistent with those recorded by Gu et al. (2023) and Hwang et al. (2016). According to other research, a collagen membrane’s ability was resisted the proteolytic attacking from the surrounding tissue lead to maintaining the space beneath and preventing the growth of fibrous connective tissues as its initial mechanical strength (Khoshkhoonejad et al., 2006). Furthermore, in contrast to the control group, the using of an acellular FSB scaffold in this investigation might be prevented ulnar osseous hypertrophy from influencing bone growth in the radius defect, this result agreed with findings of Bodde et al. (2008) and Zhao et al. (2016). A radiolucent segmental defect area of FSB-BMC treated group post –operation was observed in (Figure 6M). A slight periosteal reaction at both ends of the segmental radius defect with slight radiopaque foci in the center of the defect were seen at 2nd week post –operation (Figure 6N). The periosteal reaction at both ends of the segmental radius defect and the opacity of the foci in the center of the defect were increased at 4th week post –operation (Figure 6O). An increase in a new bone growth was observed at the six-week following surgery, and it was extended as a straight line from the proximal to the distal end of the bone defect as in (Figure 6P). The segmental bone defect was bridged by an increase in callus formation opacity in the eighth week after surgery, (Figure 6Q). The segmental bone defect was constantly continued to complete bridging of the segmental defect of radius bone by new bone formation at10th and 12th week post-operation (Figure 6R), these results might be attributed to action of acellular FSB scaffold which it was considered an essential to the guiding bone regeneration technique. (Peng et al., 2023). In addition to the action of autologous BMC that it was played an important role in healing the site of the defect. The present study showed allowing the aspirated autologous bone marrow to clot lead to producing implantable material with good handling properties to fit the site of the bone defect, this clarification was in agreement with the findings reported by Healey et al. (1990). According to Travlos (2006) and Korf-klingebiel (2008), they mentioned that bone marrow contained a variety of blood cell types, growth factors, cytokines, and stem cells. All of these cell types lead to an enhancemen of bone defect healing process. Eesa et al., (2006) and Thanoon et al., (2019) they also discovered that improving of bone marrow by these cells had a critical role in promoting and enhancing the fracture healing.

The result of radiographic results at six weeks might be related to the activation of platelets in the BMC lead to degranulation and releasing of pro-osteogenic, pro-angiogenic and pro-mitogenic growth factors such as BMP-2, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF-2) during BMA clotting (Sipe et al., 2004; Italiano et al., 2008; Kalén et al., 2008; Lim et al., 2019). Other researchers found higher levels of growth factors in BMA clot compared with peripheral blood clot. In consequently, the a raising in chemotactic response of reparative cells such as MSCs lead to enhancement the ability of various kinds of tissue cells to differentiate and multiply such as chondroblast, osteoblast and osteocyte which they were played an important role in enhancing the healing process of bone defect and this result of current study was agreed with findings reported by Fiedler et al. (2002); Crovace et al., (2008); Shoji et al. (2017); Harwood et al. (2010), Claes et al. (2012); Thanoon et al. (2019).

In general, the procedure succeed in using barrier membrane as guiding bone regeneration depended on the defect size lead to allow in a new bone formation (Hämmerle and Karring, 1998; Olaechea et al., 2016). Insufficient mechanical strength or early degradation of the barrier membrane might be caused soft tissue invasion. For this reason, a crosslinking agent was incorporated during preparation of some collagen membrane to improve its mechanical and degradation properties (Weadock et al., 1996).

Fish swim bladder (FSB) scaffolds were easily prepared from the waste product of carp fish with mild chemicals, and it was acted as a good guided for bone regeneration. In addition, the combination between FSB and autologous bone marrow clot gave a success results in bone regeneration and bridging segmental bone defect without any clinical complications related to infection or rejection. The study recommended to an additional efforts were required to optimizing the prepared FSB and determining the effectiveness of FSB in conjunction with other materials like hydrogel, platelet-rich fibrin matrix, hydroxyapatite or other sources of autologous cells in regeneration of a critical segmental bone defect.

The authors express their gratitude to the members of the surgery department at the University of Baghdad’s College of Veterinary Medicine for their assistance in this work.

The distinctive feature of this work is that it is the first to prepare fish swim bladders for local application as guided bone regeneration using autologous bone marrow clots to treat segmental bone defects. The project also aims to explore the potential of these treatments for tissue regeneration after injury.

The authors’ combined efforts have resulted in the completion of this article.

All data will be provided by the authors upon reasonable request.

The authors of this article worked on it and declared that they had no conflicts of interest.