Outcome Analysis of Osseous Ingrowth in an Artificially Created Gap Non-union Using the Novel 3D Biodegradable Polycaprolactone Poly-l-Lactide Polymer Scaffold: Insights from an Experimental Study

Abstract

Background

Synthetic biopolymers have been widely used to manage bone effects in recent years. The study aims to analyse the ability to repair artificially created ulnar bone defects with the scaffold made of Polycaprolactone (PCL) and investigate the material's feasibility as a bone graft substitute.

Method

We have tested a novel 3D biodegradable Polycaprolactone Poly-l-Lactide polymer scaffold in an experimental animal model. 14 adults New Zealand white rabbits were used to create the ulnar defect model of 10 mm in length, and randomly divided into group A (test-12 rabbits), group B (control-3 rabbits). The defect area was implanted with the PCL scaffold in the test group, whereas it was left as such in the control group. The repairing effect was observed by gross, histology, radiology, and the Scanning electron microscopy (SEM) at 4, 8, and 12 weeks. Cook's scoring was used to assess the radiological parameters.

Results

Histological and radiological results showed better quality of bone regeneration in the defect area at 12 week follow-up period. The SEM image at that period showed impregnation of the osteogenic cells in the surface and pores of the scaffold material. It was evident that the scaffold was thoroughly degraded, corresponding with osteogenesis. New bone formation was statistically significant in the test group than in the control group.

Conclusion

The Polycaprolactone Poly-l-Lactide polymer scaffold is biodegradable in-vivo at a suitable half-life. It has an excellent porous structure, no tissue toxicity, excellent mechanical strength, high osteogenesis potential, and osteoconductivity. Therefore, it can be used as bone graft material in the gap non-union and as a void filler in bone defects.

Introduction

The bone defect is a major clinical challenge in orthopaedics either caused by trauma, infection, tumour excision, and deformity correction surgeries often demand the transplantation of bone grafts or substitutes to restore bone integrity. Autologous bone grafts are currently considered the gold standard for bone defect regeneration [1]. The repair of bone defects in reconstructive surgery using autograft is subjected to significant limitations such as donor site morbidity, limited supply of autograft, additional surgical procedure, and increased blood loss. The major drawbacks of using allografts are the risk of infection, immune rejection of the allograft, and disease transmission. Demineralised bone matrix (DBM) can be produced from the allogenic bone tissue; though it reduces the risk of disease transmission, the processing and sterilisation techniques significantly decrease the therapeutic benefits of the graft [2]. Other techniques include mechanical stimulation, which is the least effective and time-consuming.

Growth factors such as BMP may require molecular carriers, and stem cell therapy requires staged technically demanding procedures; both are expensive. To overcome the drawbacks, recent research in bone tissue engineering focussed on biological materials and their composites in treating bone defects [3]. The ideal bone graft should possess osteogenic, osteoinductive, and osteoconductive, and provide structural support. However, the development of ideal bone fillers is remained a significant challenge in clinical practise, as the previous study material like bio-ceramics including hydroxyapatite, tricalcium phosphate, silicate ceramics, mesoporous bioactive glass made of silica, phosphate, etc. lack properties of biodegradation, mechanical strength, osteoconductivity, etc. [4].

Amongst many types of biomaterials, synthetic biodegradable polycaprolactone is widely used in the field of ortho-biologics [5]. Because of its biodegradability, enhanced porosity creates the space for tissue regeneration, excellent biocompatibility, and good mechanical strength; the scaffolds are of most interest for its clinical applications [6].

Materials and Methods

Selection of Animals

The study was conducted during September between 2017 and 2019, and the Institutional Ethical Committee approved all animal procedures. The subjects of the experimental study were 15 adult New Zealand rabbits (Oryctolagus cuniculus) of average 2.5–3.0 kg, which were chosen and left for 2 weeks before starting the experiment [7].

The principle of laboratory care, feeding, and sacrifice of animals was followed as per the ICMR guidelines and CPCSEA guidelines on the care of experimental animals with ethical clearance from Central Animal Ethical Clearance Committee of 542/02/ab/CPCSEA dated 27.12.2012 with approval no. 2017/CAEC/717. This study involves sacrificing the animal at regular intervals; hence, the predicted outcome of the non-interventional group (the results of similar experimental studies in our institute [8, 9]) is kept minimum because of the lack of resources as advised by the institutional ethical committee.

Preparation of PCL (Polycaprolactone) Scaffolds

PCL solutions with concentrations of 10% (w/v) and 15% (w/v) were prepared by dissolving PCL in tetrahydrofuran (THF) at 50 °C for more than 2 h. The two different solutions were mixed with a magnetic stirrer at 300C (6 h) to obtain homogeneity and to get a critical performance of the solution in a bioreactor. Then, the solution was transferred into glass Petri dishes (5 cm in diameter and 1.5 cm in depth). All of the samples were frozen at − 80 °C for 4 days. Then, they were transferred into freeze-drier (Christ, Germany) and lyophilised at − 80 °C for 2 days to ensure that they were completely dried.

Fourier Transform Infrared spectroscopy (FTIR)

The chemical structures of the scaffolds were investigated using Fourier Transform Infrared spectroscopy. Tablet samples were prepared with dried PCL samples dispersed in KBr. The spectra of samples were taken at 400–4000 cm⁻¹ wavelength with FTIR-8101 (Shimadzu, Tokyo, Japan).

Scaffold Wettability and SEM

The scaffold wettability was assessed with water contact angle values by the sessile drop method at room temperature (Kruss DSA 100, Germany). Surface and cross-section morphologies of lyophilised PCL scaffolds before and after degradation were examined using an SEM (scanning electron microscopy) (Leo 435 VP, UK) after coating with a gold–palladium layer shows highly interconnected fibres of different pore sizes as in Fig. 1.

Fig. 1.

The scanning electron microscopic image of the prepared PCL scaffold shows the micropores of different sizes (asterisks), the higher degree of interconnectivity (arrowhead), and the isotropic tubular morphology (arrow)

Mechanical Testing and Biodegradability

The mechanical strength of the material is essential for its practical applications. The uniaxial compression machine delivers a kilogram-force per centimetre square (kgf/cm²)/98.0665 kPa (kilopascals) called as the technical atmosphere (symbol: at), the stress–strain curves of the PCL polymer scaffold under this uniaxial compression test showing a typical foam behaviour and low modulus of elasticity of the materials, as shown in Fig. 2. The percentage weight loss was considered the measure of biodegradation during that particular period after the enzyme treatment under certain conditions, as shown in Fig. 3.

Fig. 2.

a Compression behavior experiment of the polymeric scaffolds in a universal testing machine (Instron-Series 5548, Germany. b The stress–strain curves of the PCL scaffold under a uniaxial compression test showing a typical foam behavior of the materials (low modulus of elasticity)

Fig. 3.

Percentage weight loss of the polymer scaffold by Pseudomonas cepacia lipase (0.5 mg/ml) at 37 °C

Operative Procedure in Rabbits

Fifteen adult rabbits were used. The surgery was performed under anaesthesia using ketamine injection intramuscularly (30 mg/kg). The forearm was shaved and disinfected with spirit and povidone-iodine in each rabbit. The mid-diaphysis of the ulna was reached through proper dissection. Then a gap of 10 mm was created in each rabbit. Bone debris was washed out using normal saline. Out of fourteen rabbits, 2 rabbits were made control in which after no scaffold was inserted in the created gap, and 12 rabbits were made experimental in which the scaffold (polycaprolactone) was inserted into the created gap without external fixation, as shown in Fig. 4. The surgical wound was closed in layers under aseptic precautions. They were followed up for 4, 8, and 12 weeks.

Fig. 4.

A (i), (ii), (iii), (iv) showing the gross images of impregnated PCL scaffold in the defect and the formation of new bone at 12 weeks. (v) Shows the resected portion of the ulna with newly formed bone tissue in the defect area. B Showing the clinical images of wound healing at 4, 8 and 12 weeks intervals

Gross and Radiological Assessment

The gross examination of the operative site was done to evaluate the degradability of the original scaffold, the morphology and texture of the newly formed tissue, and the continuity of the newly formed bone with the host bone at regular intervals. Sequential radiographic images were captured at 4, 8, and 12 weeks after surgery. Images were captured at 50 kV and at 4 mAs. The degree of new bone formation was assessed using Cook's criteria [10] are as follows: 0—no visible new bone formation, 1—minimal new bone formation visible haphazardly, 2—scaffold-host bridging bone formation at both the ends, 3—organised new bone with improved cortical intensity at the margins, 4—the disappearance of scaffold-host distinction, and 5—significant bone growth with gap filling and remodelling.

Histological and SEM Assessment

Resected sample specimens (operated part) were collected in 10% formalin and kept for fixation for 12–24 h. Then, the samples were decalcified (distil water 82 ml + formic acid 8 ml + 10 ml HCL). After completing the decalcification process, the representative samples were processed in the automatic Histokinette machine (Leica); S1 No- 514; Year-26.04.2016. The paraffin blocks were sectioned into 5 μm thickness using Paraffin Embedding System (Leica); Sl No-515; Year-26.04.2016. All the samples were stained with H&E, and it was assessed for osteocyte colonisation. The SEM evaluation at 12 weeks shows osteogenesis, cell proliferation, scaffold integration, and adherence to the host bone, as shown in Fig. 6D.

Fig. 6.

A and B Histological staining of the control and test specimen of the ulna at 4, 8 and 12 weeks in the defect. C Shows the quantitative data from A and B. The results revealed osteogenesis in the test group then in the control group. HB Host Bone, NB New Bone, F Fibrous tissue. D Shows SEM images, Arrow head appearance of osteoblast in the scaffold background with porous architecture, Asteris scaffold-osteoblast integration and proliferation, Arrow colonization and growth of osteoblast with disappearance of the scaffold

Statistical Analyses

The mean radiological score at different intervals was calculated in both groups. The quantitative variables were evaluated by two-tailed Welch's unequal variances t test to assess for significant differences between these two groups. The P value of < 0.05 was considered statistically significant.

Result

There were a total of 15 rabbits operated on and observed in this study. These rabbits have been divided, so that there were 3 rabbits in the control group and 12 rabbits in the test group. They were followed up for 4, 8, and12 weeks. They were sacrificed and assessed by clinical, radiological, gross, histological examination, and scanning electron microscopy at those regular intervals.

On clinical examination of the operative site during the follow-up at 4, 8, and 12 weeks, there is gradual healing of the wound and no foreign body reaction due to scaffold-like gapping of the wound, infection at the site of operation, and no immunological reaction like erythema and local oedema. On gross examination at autopsy at 4 week interval, there were no signs of infection like pus formation and no inflammatory reaction to the surrounding tissue like necrosis of muscle tissue, etc. As the weeks increase, there is good adhesion between the bone and scaffold interface and the scaffold was not displaced and enclosed in fibro callus tissue; there is also a gradual decrease in the size of the scaffold, seen by naked eyes. It shows the gradual degradation of polycaprolactone scaffold and the new bone formation in the defect site. Further, it shows the newly formed bone is continuous with the host bone.

The radiographs showed apparent defects at the implant sites at week 4. However, progressive low-density developments were also seen in the ulnar defect areas in the subsequent weeks. At 12 weeks, defects were still seen in the Control animal, whilst in PCL scaffold areas, the defects were filled in with a new bone, and found to be statistically significant, as shown in Fig. 5.

Fig. 5.

Radiographs showing no bone regenaration in the control group and different stages of newly formed bone in the test group from 4 to 12 weeks

The histologic and SEM study was performed at 4, 8, and 12 weeks to evaluate the new bone formation with data shown in Fig. 6. At 4 weeks, there were predominant inflammatory cells evident around the scaffold, and there was fibrous tissue formation in both groups. At 8 weeks, only fibrous connective tissue proliferation was seen in the control group, whereas in the PCL scaffold group, the scaffold materials partially disappeared, and new bone was observed. At 12 weeks, most of the area of bone defect was covered by fibrous connective tissue in the control group, indicating that new bone had not been formed, whereas in the PCL group, the scaffold was completely degraded, and the defect was obliterated completely, suggests new bone formation.

Discussion

Bone grafts are essential to provide structural support, filling defects, and enhance the osteogenesis of the affected bone. Scientific studies showed fresh autologous bone is the most suitable and effective graft material in various clinical situations [11]. However, autologous bone has several significant disadvantages: (1) the available supply of autologous bone grafts is often insufficient for more extensive defects, particularly in children; (2) the donor site problems like pain, bleeding, dehiscence, cosmetic deformity, infection, and nerve injury; and (3) the ability to fabricate a graft with exact morphology of the recipient area is often limited, resulting in less optimal filling of the defect [12].

Despite the increase in the number of procedures that require bone grafting, there has not been a single ideal bone graft substitute. The other suitable alternatives to autologous bone are homologous bone (allografts), heterologous bone (xenografts), demineralised bone, and synthetically derived organic and inorganic substances. Nevertheless, these alternatives have been largely ineffective in fulfilling bone regeneration's basic mechanisms: vis-a-via osteogenesis, osteoinduction, and osteoconduction. Biomedical scientists and surgeons, thus, have a tremendous responsibility to develop biological alternatives that will enhance the functional capabilities of the bone graft substitute and potentially reduce or eliminate the need for autograft [13]. In skeletal tissue engineering, experimental work on the rabbit diaphyseal defect bone model has been well studied in assessing numerous properties of biomaterials [7].

Under the conditions used in the present study, the implantation of the polycaprolactone scaffold leads to the formation of new bone in the critical size defect area. It is established in this study that the newly formed bone is uniformly distributed and integrated with the host bone.

Radiological examination of ulna revealed progressive resorption of the PCL scaffold and some new bone formation; the gross examination of the specimens substantiated this finding. Previously similar studies using bioactive glass ceramics, carbon fibreglass implants, fibre-composites, and various other ceramic implants did not show any change in the consistency of the implant even after 20 weeks of follow-up, since none of the implants was biodegradable [7, 14, 15]. In those previously described biodegradable implants, the resorption rate was found to be higher (resorption of the implant was not associated with corresponding new bone growth). This discrepancy could be assumed to be due to early degradation of the scaffold when compared to the new bone formation, which requires a longer duration [17].

The design and selection of an ideal carrier for the delivery of osteoblast cells are, the material should provide uniform loading, a cell–cell anchorage (osteoconductive bridging of host bone by the new bone), rapid vascular ingrowth, and biodegradability at appropriate period [18].

The in-vivo study done by Lee et al. on rabbits with a 5 mm diaphyseal defect in the left radius showed adequate new bone formation only in the composite PCL (PCL + βTCP and PCL + duck beak) scaffold [19]. To overcome the drawbacks, in this study, we have improved the microporosity, the compressive strength, and the degree of cross-linking without adding the additives. Also, it makes the manufacturing process less expensive. Moreover, the critical size of the defect evaluated in this study is about 10 mm, so it is possible to deal with the larger bone defects. The other in-vitro study by Zheng et al. proved that the mesenchymal stem cell (MScs-harvested from the distal femur of the experimental animal using microfracture) showed better adhesion, proliferation, and improved osteogenic differentiation. The regenerative potency of the MSCs had shown to be enhanced when it was augmented with the PCL-HA scaffold [20].

Previous studies have suggested that a small pore size improves bone growth into scaffolds and provides attachment points for osteoblasts [21]. The nanofiber matrices follow a low erosion rate PGA > PLGA > PLLA > PCL [22].

This study fabricates a synthetic biodegradable bone scaffold with improved mechanical properties and excellent biocompatibility. The porous polycaprolactone scaffold has attached much attention as bone substitutes because of its ability to lead osteoconductivity and adequate mechanical strength, which proved in-vitro studies. The present studies described a freeze-drying technique to fabricate polymer scaffolds with high porosity and controlled pore architecture. The microarchitecture of the scaffold has been satisfactorily obtained using different solvent systems.

Conclusion

In the present study, we developed a novel PCL scaffold. Gross, X-ray, and histological analyses which revealed the scaffold promotes new bone formation in rabbit ulna by promoting osteogenesis and osteoconduction. It shows that PCL scaffold may be a good option for the treatment of non-union.

Declarations

Conflict of interest

Author declares no potential conflict of interest.

Ethical standard statement

This article does not contain any studies with human or animal subjects performed by the any of the authors.

Informed consent

For this type of study informed consent is not required.