3D-printed Poly-Lactic Co-Glycolic Acid (PLGA) scaffolds in non-critical bone defects impede bone regeneration in rabbit tibia bone

Abstract

BACKGROUND:

An increasing number of bone graft materials are commercially available and vary in their composition, mechanism of action, costs, and indications.

OBJECTIVE:

A commercially available PLGA scaffold produced using 3D printing technology has been used to promote the preservation of the alveolar socket after tooth extraction. We examined its influence on bone regeneration in long bones of New Zealand White rabbits.

METHODS:

5.0-mm-diameter circular defects were created on the tibia bones of eight rabbits. Two groups were studied: (1) control group, in which the bone defects were left empty; (2) scaffold group, in which the PLGA scaffolds were implanted into the bone defect. Radiography was performed every two weeks postoperatively. After sacrifice, bone specimens were isolated and examined by micro-computed tomography and histology.

RESULTS:

Scaffolds were not degraded by eight weeks after surgery. Micro-computed tomography and histology showed that in the region of bone defects that was occupied by scaffolds, bone regeneration was compromised and the total bone volume/total volume ratio (BV/TV) was significantly lower.

CONCLUSION:

The implantation of this scaffold impedes bone regeneration in a non-critical bone defect. Implantation of bone scaffolds, if unnecessary, lead to a slower rate of fracture healing.

Keywords

Bone substitute bone graft bone defect Poly-Lactic Co-Glycolic Acid

1. Introduction

Bone graft procedures are commonly done to treat bone defects. The problems of bone grafting include the availability of graft material, donor-site morbidity, immunogenicity and biomechanical integrity [1]. An increasing number of bone graft materials are commercially available. These materials vary in their composition, mechanism of action, costs, and as a result, indications [2,3].

Bioscaff^TM Alvelac^TM (Bio-Scaffold International Pte Ltd, Singapore), is a commercially available scaffold produced for dental applications. Specifically, it is being used for preservation of the alveolar tooth socket after tooth extraction. It is a polymer of lactic acid and glycolic acid (Table 1), and was produced using 3D-printing technology. This product was designed with macro-channels and micro-pores for cell adhesion and nutrient flow as well as to provide necessary space for bone tissues to grow. Previous studies had shown that PLGA does not induce any inflammatory reaction or damage to the local tissue, and is one of the most popular biodegradable polymer [4,5].

Table 1
Properties of the PLGA scaffold [9]

Material composition Lactic acid: Glycolic acid (85:15)

Average pore size 20–150 μm

Volumetric porosity 70%

Sterilization Dry heat

Strength 6 MPa

Typical degradation time after placement 2–6 month

Material composition	Lactic acid: Glycolic acid (85:15)
Average pore size	20–150 μm
Volumetric porosity	70%
Sterilization	Dry heat
Strength	6 MPa
Typical degradation time after placement	2–6 month

The efficacy of this scaffold in long bone remains unknown. The aim of this study is to examine the influence of 3D printed PLGA scaffold on bone regeneration in a rabbit bone defect model, looking at the rate of bone regeneration and the associated tissue reaction to this scaffold.

2. Materials and methods

2.1. Animal model

All experimental procedures and protocols used in this study were reviewed and approved by our Institutional Animal Care and Use Ethics Committee. Eight female New Zealand White rabbits (weighing 2.5 kg–3 kg) were used. The rabbits were anaesthetised with an intramuscular injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Anesthesia was then maintained with isoflurane during surgery. Both hind legs of each rabbit were randomly distributed into two groups: (1) control, in which the bone defects were not treated; (2) scaffold group, in which the PLGA scaffolds (diameter 4 mm, depth 5 mm) were implanted into the bone defect. A 2 cm longitudinal skin incision was made on the medial side of the tibia bone. The implant sites (diameter 5 mm) were prepared using an electric drill after exposing the cortical bone. The location of bone defect was marked by inserting two Kirschner-wires 2 mm apart from the edge of the defect. The scaffolds were secured in position by suturing the muscle and skin in layers over the bone defect. After surgery, the animals were allowed to move freely within the cage. Radiographs of the tibia bone were taken at 2 weekly intervals. The rabbits were sacrificed at either 4 or 8 weeks (4 rabbits each time) after surgery and the tibia bones analysed using micro-computed tomography (micro-CT) and histology.

2.2. Radiography

Radiographs were taken of the operated hind limb every two weeks postoperatively to assess for new bone formation in the bone defects and to monitor for any post-operative complications.

2.3. Micro-CT analysis

New bone formation in bone defects were analysed with a Quantum FX micro-CT (PerkinElmer, USA). Each tibia bone was placed in a 50 ml Falcon tube for the scan. Scans were obtained with X-ray energy at 90 kV and 160 μA with a 360° rotation. Skyscan CT Analyser software was used to analyze the data from the scan. The bone defects were marked as the region of interest. The architectural parameters obtained from the binarised volumes of interest was bone volume fraction (BV/TV). TV is the volume of the whole bone defect, BV is the volume of the new mineralised tissue formed during bone healing, and BV/TV is the relative bone volume by normalizing the volume of mineralised tissue in the defect by the total volume of the bone defect.

2.4. Histology

Tibia bones were isolated then processed by fixing in 10% formalin neutral buffer solution, decalcified in formic acid, and then embedded in paraffin. The samples underwent histological examination with hematoxylin eosin staining under a light microscope. The purpose of this analysis was to examine the quality of new bone formation as well as to evaluate for any foreign body reaction to the scaffold.

2.5. Statistical analysis

Data were presented as means (standard deviation). Error bars in all figures represent standard deviation. The results were analysed using the Student’s t-test.

3. Results

There were no signs of infection or other post-operative complications in all rabbits, and all animals survived. Eight weeks after surgery, the PLGA scaffolds were still present at the site of the bone defects.

At two weeks after surgery, the radiograph showed a radiolucent zone of circular bone defect in tibia bones (Fig. 1). The radiolucent zone has a clear margin. From four to eight weeks after surgery, the radio density in the radiolucent zone gradually decreased in the control group. In comparison, radiolucent zone of bone defect appeared equally visible eight weeks after surgery in the scaffold group.

Fig. 1.

Radiographic photos at defect site, taken after 2 weeks, 4 weeks, 6 weeks and 8 weeks postoperatively for control group and scaffold group in the same rabbit. The defect in the control group gets less radio-opaque over time, in contrast to the defect in the scaffold group.

Micro-CT images showed that the bone defect was gradually filled with newly formed bone in the control group four weeks and eight weeks after surgery (Fig. 2). In contrast, new bone formation was not observed in the areas that were occupied by PLGA scaffolds. Accordingly, the total bone volume/total volume ratio (BT/TV) in the control group was significantly higher than in the scaffold group as shown in Fig. 3. The micro-CT analysis thus demonstrated that there is more bone formation in the control group compared to the scaffold group.

Fig. 2.

Micro-CT analysis at 4 and 8 weeks after surgery. (A) Area marked in red was analyzed using a computer software. (B) Comparative micro-CT images of control group and scaffold group at 4 weeks after surgery. (C) Comparative micro-CT images of control group and scaffold group at 8 weeks after surgery.

Fig. 3.

Bone Volume/Total Volume (BV/TV) values of control group and scaffold group at 4 and 8 weeks post-surgery. P value was <0.05 for both 4 and 8 weeks.

PLGA scaffold embedded in paraffin was shattered during sectioning with a microtome and an empty space was left in the bone defect on histology sections. New bone formation was observed in both control group and scaffold group. Eight weeks after surgery, it was evident that there was more new bone formation in the control group compared to the scaffold group, as shown in Fig. 4. The fact that there was new bone bridging across the gap in the control group shows that the defect is non-critical. In the scaffold group, it was noted that bone grows over and bypasses the scaffold, instead of growing into it. No foreign body reaction was noted in the histology specimens in the scaffold group.

Fig. 4.

Representative images of cross sectional sections of histological analysis of bone healing at the site of defects at eight weeks after surgery. Hematoxylin-Eosin staining demonstrates new bone (NB) formation. Magnification: 40X. (A) Control group. New bone grew across the defect. (B) Scaffold group. The scaffold was shattered during sectioning and left an empty space in the bone defect. New bone is seen to bypass the scaffold.

4. Discussion and conclusion

The ideal synthetic bone graft should be osteoconductive, osteoinductive, osteogenetic, and biocompatible, without the risk of transmitting infectious diseases [6]. It should also have similar strength to native bone, and be cost effective. Most bone graft substitutes available in the market are osteoconductive in nature [7,8], unless bone morphogenic proteins were added. They differ in their mechanical strength, porosity and degradation time. There is a fine balance between speed of degradation and loss of mechanical strength. With a shorter degradation time, the scaffold will lose its function as a mechanical support earlier. Hence, the choice of material depends on the indication for bone grafting – as a bone void filler, bone graft extender or bone graft substitute. The properties required for each of these indications are different, and as such, choosing the correct bone graft substitute is crucial in achieving bone union.

It has been demonstrated that porosity and pore size of biomaterial scaffolds play a critical role in bone formation both in vitro and in vivo. Based on previous studies, the minimum pore size is around 100 μm to fulfil cell size and migration requirements [9]. Porosity is important for osteoblasts to utilise the scaffold as a framework to spread and generate new bone, and graduated pore sizes have been reported to promote better cartilage and bone formation [10]. According to manufacturer’s information [11], the average pore size of this scaffold is between 20–150 μm and the volumetric porosity of this scaffold is around 70%. There is an upper limit in porosity and pore size limited by constraints associated with mechanical strength. New fabrication techniques, such as 3D printing, enabled customization of scaffold for specific morphology and mechanical properties more easily than conventional methods [12,13], and has been utilised in biofabrication [14].

This study showed that there is a lack of bone growth through the scaffold when compared to control, as evidenced by the radiographic appearance, BV/TV ratio and histological findings. The PLGA scaffolds failed to degrade at the end of the study period, and new bone bypasses the scaffold and grows superficial to it, instead of utilizing the scaffold as a framework to grow across the defect. 8 weeks was chosen as the total study duration as one would expect healing with hard callus formation in a non-critical defect at that point in time. However, instead of behaving as an osteoconductive scaffold, the presence of this scaffold seemed to have blocked early new bone formation, which will be detrimental in fracture healing. Similarly, it was reported that composite implant of bovine bone morphogenetic protein and biocoral failed to improve healing in the treatment of scaphoid nonunion [15].

The clinical efficacy of synthetic bone replacement material needs careful evaluation, as composition, material and structural properties, and perhaps even manufacture and storage could have effects on the actual ability of the product to positively influence bone healing. When not used in the appropriate clinical scenario, bone graft substitutes may impede bone healing. Even though the use of this scaffold has been shown to impede bone healing in a non-critical defect, there is still interest in studying the use of this scaffold as the manufacture process and the material used has greatly reduced the cost of this scaffold compared to other bone replacement material available in our practice. Future research can be geared towards looking at the healing of critical size bone defect with the use of this PLGA scaffold.

Footnotes

Conflict of interest

None to report.

References

Younger

E.M.

and Chapman

M.W.

, Morbidity at bone graft donor sites, J Orthop Trauma 3 (1989), 192.

Pryor

L.S.

Gage

Langevin

Herrera

Breithaupt

A.D.

Gordon

C.R.

, Review of bone substitutes, Craniomaxillofac Trauma Reconstr 2(3/4) (2009), 151.

Giannoudis

P.V.

Dinopoulos

and Tsiridis

, Bone substitutes: An update, Injury 36S (2005), S20.

Ulery

B.D.

Nair

L.S.

and Laurencin

C.T.

, Biomedical applications of biodegradable polymers, J Polym Sci B Polym Phys 49(12) (2011), 832.

Pan

and Ding

, Poly(lactide-co-glycolide) porous scaffolds for tissue engineering and regenerative medicine, Interface Focus 2 (2012), 366.

Lane

J.M.

Tomin

and Bostrom

M.P.G.

, Biosynthetic bone grafting, Clin Orthop Relat Res 367S (1999), S107.

Calori

G.M.

Mazza

Colombo

and Ripamonti

, The use of bone-graft substitutes in large bone defects: Any specific needs? Injury 42 (2011), S56.

Ashman

and Phillips

A.M.

, Treatment of non-unions with bone defects: Which option and why? Injury 44(S1) (2013), S43.

Karageorgiou

and Kaplan

, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials 26 (2005), 5474.

10.

Woodfield

T.B.

Blitterswijk

Wijn

Sims

T.J.

Hollander

A.P.

and Riesle

, Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissueengineered cartilage constructs, Tissue Eng 11(9–10) (2005), 1297.

11.

Bio-Scaffold International Pte Ltd. Product Data Sheet. Available at: http://www.bio-scaffold.com/web/wp-content/uploads/2009/09/web_ProductDataSheet.pdf. Accesssed July 30, 2016.

12.

Jin

and Cao

, Manufacture of degradable polymeric scaffolds for bone regeneration, Biomed Mater 3 (2008), 022001.

13.

Yeong

Chua

Leong

and Chandrasekaran

, Rapid prototyping in tissue engineering: challenges and potential, Trends Biotechnol 22(12) (2004), 643.

14.

Arealisa

and Nikolaoub

V.S.

, Bone printing: New frontiers in the treatment of bone defects, Injury 46(S8) (2015), S20.

15.

Kujala

Raatikainen

Ryhanen

Kaarela

and Jalovaara

, Composite implant of native bovine bone morphogenetic protein (BMP) and biocoral in the treatment of scaphoid nonunions—A preliminary study, Scand J Surgery 91 (2002), 186.