Preparation of vancomycin-loaded gelatin/β-TCP composite scaffolds

The scaffolds were prepared by a previously reported freeze-casting method with minor modifications.¹⁸ Scaffolds were typically obtained as follows: briefly, a gelatin aqueous solution of 16.67 weight% was prepared by dissolving 2.5 g of gelatin powder Type A, G-2500, approximately 300 Bloom, (Sigma-Aldrich, St. Louis, Missouri) into 15 mL of deionized water at 50°C with stirring for one hour. Subsequently, different amounts of β-TCP (0 g, 0.25 g, 0.75 g and 1.25 g) were added to 15 mL of gelatin solution at 37°C to obtain suspensions with different compositions. The ratios between β-TCP and gelatin (weight/weight %) were 0:10, 1:10, 3:10 and 5:10. After constant stirring for 45 minutes, 2 mL aqueous solution containing 250 mg of vancomycin (one-tenth of the total amount of gelatin) and 8 mL aqueous solution containing 0.234 g of gelatin (one-64th of the total amount of gelatin) were added to the gelatin/β-TCP composite solution. After the crosslinking reaction was carried out at room temperature for 48 hours, the crosslinked gelatin hydrogels were frozen at -20℃ for 24 hours and then freeze-dried. The composite scaffolds were labelled according to their contents in the inorganic phase as follows: G-TCP0 (the weight ratio between β-TCP and gelatin was 0:10), G-TCP1(the weight ratio between β-TCP and gelatin was 1:10), G-TCP3 (the weight ratio between β-TCP and gelatin was 3:10) and G-TCP5 (the weight ratio between β-TCP and gelatin was 5:10). The acquired scaffolds were then sputter-coated with gold and the morphology was examined under a scanning electron microscope (SEM), JSM-5600, (JEOL CO. LTD, Tokyo, Japan). The mean pore size of the scaffolds was evaluated by geometrical measurements on the scanning electron micrographs, and the porosity of the composite scaffold was measured by the method reported previously.¹⁹ In brief, the porosity of the pure gelatin scaffold, εgelatin, was calculated from its apparent density, ρ*gelatin, and the density of gelatin, ρgelatin (1.25 g/cm³), by the following equation: εgelatin = 1 −ρ*gelatin/ρgelatin, where ρ*gelatin was determined by measuring the weight and volume of a cubic pure gelatin scaffold. The porosity of the gelatin/β-TCP composite scaffold, εC, was calculated by the following equation: εC = εgelatin − ρ*C/ρC, where ρC was the density of β-TCP (3.14 g/cm³) and ρ*C was the apparent density of the hybrid sponge which was derived from the weight and volume of the cubic hybrid sponge.

The release profile and antibacterial properties of vancomycin released from composite scaffolds

The release profile of vancomycin released from the composite scaffold was identified using the Kirby-Bauer (K-B) disc diffusion method.

The standard curve was first established to explore the optimal drug concentration by inputting the diameters of inhibition zones. Filter paper discs (diameter 8 mm) impregnated with 20 μL of serial vancomycin dilutions of known concentrations were aseptically placed on petri plates (Thermo Fisher Scientific Inc, Waltham, Massachusetts) containing Mueller-Hinton agar (Difco) (Thermo Fisher Scientific Inc,) and seeded with 10⁸ colony-forming units (CFU)/mL (0.5 McFarland)²⁰ of Staphylococcus epidermidis strain RP62A and then the petri plates were incubated overnight. The diameters of the inhibition zones around the paper discs were determined with a micrometer. The correlation between zone sizes and vancomycin concentrations (150 μg/mL, 100 μg/mL, 80 μg/mL, 60 μg/mL, 40 μg/mL, 20 μg/mL and 10 μg/mL) were identified by constructing a standard curve. The zone diameter for each test sample was determined as a mean of six inhibition zones in two agar plates (three discs per plate). Regression analysis was performed with Microsoft Excel 2016 (Microsoft Corp., Redmond, Washington) to generate the final standard curve.

A total of 27 nine-week-old Sprague Dawley rats, each weighing 250 g to 300 g, were obtained, and each rat was implanted with one scaffold from each group, totaling four scaffolds per rat (G-TCP0, G-TCP1, G-TCP3 and G-TCP5). Before implantation, the gelatin/β-TCP composite scaffolds were cut into a cube (5 mm × 5 mm × 5 mm in size, the weight of each implant was approximately 30 mg) and sterilized with ethylene oxide. Chloral hydrate (Wako Pure Chemical Industries, Ltd, Hokkaidō, Japan) served as an anaesthetic agent (peritoneal injection, 3.0 mg/100 g body weight). Under sterile conditions, four muscular pouches were established on the back of each rat, where vancomycin-loaded scaffolds were implanted. At each of the designated timepoints (three days, seven days, ten days, 14 days, 21 days, 28 days, 35 days, 42 days and 56 days), three rats were sacrificed. An amount of 1.0 g of muscle specimen close to the implants (0.5cm apart around the center of the specimen) was collected and homogenized, and then the obtained homogenate was resuspended in 5 mL of physiological saline to measure the drug concentration by the K-B method as previously described.²¹ Briefly, 20 μL of the homogenate was added onto filter paper discs in petri plates and incubated at 37°C overnight, and the inhibition zone of bacterial growth around the discs was analysed.

Preliminary biocompatibility

Intramuscular implantation is an important step to test in vivo biocompatibility and the degradation properties of a bioabsorbable polymer.²² To evaluate the biocompatibility of the vancomycin-loaded composite scaffolds, the implants in each group, along with the surrounding tissues, were removed to investigate the tissue reaction, inflammation and degradation at each of three designated timepoints (two weeks, four weeks and eight weeks after implantation). The samples harvested at different timepoints were washed in phosphate-buffered saline (PBS) and fixed in 10% formalin solution. After dehydration through a graded series of alcohols, paraffin-embedded samples were cut into 5 μm sections. Sections were then deparaffinized, rehydrated through a graded series of alcohols and stained with haematoxylin and eosin (H&E) for histological observation.

Osteomyelitis model

A total of 43 New Zealand white rabbits (21 female, 22 male), each weighing 1.8 kg to 2.2 kg (approximately six months of age), were used for this study. The osteomyelitis model was established as previously described.²³ Briefly, anaesthesia was initiated by intravenously injecting 3% sodium pentobarbital (30 mg/kg). The right hind leg of the animals was shaved and disinfected with 2% iodine solution. After being draped with sterile sheets, a 12-gauge needle was percutaneously inserted into the medullary cavity from the lateral aspect of the proximal tibial metaphysis, and 0.3 mL bone marrow was extracted. Subsequently, 0.1 mL 5% morrhuate sodium and 0.1 mL MRSA (1 × 10⁹ CFU/mL) were injected through the same needle. The needle was then flushed with 0.1 mL of 0.9% sodium chloride to ensure that all bacteria reached the tibial cavity. During the experiment, the animals were kept in individual cages, fed a routine diet and allowed to be fully active. The infection could progress for three weeks, during which the severity of osteomyelitis was radiologically determined.

The severity of osteomyelitis was confirmed by radiographs according to the descriptive criteria of Norden²⁴ at three weeks after the induction of osteomyelitis. These criteria are based on the presence of sequestra, periosteal new bone formation and destruction of bone. A numerical score was assigned to each variable. All scores were added to create a composite radiological score with a maximum of six, representing radiological severity. Two radiologists (JZ and HZ) blindly and independently evaluated these images. Three rabbits with signs of osteomyelitis on the radiograph were randomly selected and sacrificed. Gross examination and histological observation were performed to evaluate the presence of osteomyelitis.

Debridement and implantation of vancomycin-loaded composite scaffolds

The remaining 40 rabbits with radiological evidence of osteomyelitis underwent debridement at three weeks after the induction of osteomyelitis. A 3 cm incision was made over the lateral aspect of the proximal tibial metaphysis, parallel to the tibial shaft. The necrotic, hardening and infected soft and bone tissues were removed. After the infected soft and bone tissues were cleaned, the wounds were washed with 2% hydrogen peroxide solution and saline. After debridement, all of the rabbits were randomly distributed into four groups as follows: control group (n = 4) in which the rabbits were treated with debridement only ; G-TCP0 group (n = 12) in which the rabbits were treated with debridement and implanted with vancomycin-loaded G-TCP0 scaffold; G-TCP1 group (n = 12) in which the rabbits were treated with debridement and implanted with vancomycin-loaded G-TCP1 composite scaffold; and G-TCP3 group (n = 12) in which the rabbits were treated with debridement and implanted with vancomycin-loaded G-TCP3 composite scaffold. The rabbits of G-TCP0, G-TCP1 and G-TCP3 groups were implanted with the same amounts of the sterilized scaffolds (G-TCP0, G-TCP1 and G-TCP3, 1.0cm×0.5cm×0.5cm, respectively) in the defect site. After implantation, the incision was closed layer by layer.

Assessment of therapeutic efficacy: radiographic and histological evaluation

At the preset timepoints (four weeks and eight weeks after debridement), radiographic examination and histological observation of two rabbits from the control group and six rabbits from each of the other groups were conducted. After the rabbits were sacrificed, anteroposterior and lateral radiographs of their right tibias were taken at 50 kV, 100 mA and 12 ms. These images were also blindly and independently evaluated by two radiologists according to the criteria described by Norden²⁴ A numerical score was assigned to each variable. All scores were added to create a composite radiological score with a maximum of six, representing radiological severity.

After radiograph examination, the specimens from the bone defect sites were fixed with 10% paraformaldehyde, decalcified with sodium formate and embedded with paraffin. Cross-sections of bone (5 mm thickness) were stained with H&E and then examined under a light microscope. Slides were randomized and scored by an independent pathologist. Each slide was scored using the method described by Smeltzer et al²⁵ to rate signs of infection. This score consists of four categories of investigation: acute intraosseous inflammation; chronic intraosseous inflammation; periosteal inflammation; and bone necrosis. A numerical score was assigned to each variable and all scores were added to create a composite histopathologic score with a maximum of 16, representing histopathologic severity.

Statistics

All data were reported as means ± sd unless otherwise stated. A paired t-test (when the data had equal variance) and a non-parametric t-test (when the data had unequal variance) were performed using SPSS software, version 11.0 (IBM, Armonk, New York), and p < 0.05 was considered statistically significant.

Characterisation of vancomycin-loaded composite scaffolds

Figure 1 shows the electron microscopy structure of the composite scaffolds in the inorganic phase. The prepared gelatin/β-TCP scaffolds exhibited a homogeneously interconnected 3D porous structure. Moreover, the β-TCP granules presented uniform distribution on the walls of the composite scaffold. Figure 2 lists the mean diameters of the pores and porosity. The pore size of different scaffolds was not affected by the presence of β-TCP granules. However, the β-TCP granules could modify the structure of composite scaffolds and improve the porosity and interconnection. The porosity and interconnection were significantly increased in G-TCP1 and G-TCP3 scaffolds compared with the pure gelatin scaffold. In contrast, extremely high content of β-TCP granules decreased the porosity and interconnection. In addition, the porosity and interconnection were significantly decreased in the G-TCP5 scaffold.

Fig.

The prepared gelatin/β-tricalcium phosphate (β-TCP) scaffolds exhibited a homogeneously interconnected 3D porous structure. The β-TCP granules presented uniform distribution on the walls of G-TCP1, G-TCP3 and G-TCP5 composite scaffolds. a) G-TCP0 scaffold; b) G-TCP1 scaffold; c): G-TCP3 scaffold; d) G-TCP5 scaffold. Images on the right hand side are the enlarged scale of the view on the left.

Fig.

The pore size and porosity of the gelatin/β-tricalcium phosphate (β-TCP) composite scaffolds. a: The pore size of different scaffolds was not affected by the presence of β-TCP granules. (*There were no significant differences among the four different scaffolds p > 0.05, paired t-test). b: The β-TCP granules could improve the porosity of the composite scaffolds. The porosity was significantly increased in G-TCP1 and G-TCP3 scaffolds compared with the pure gelatin scaffold. In contrast, extremely high content of β-TCP granules would decrease the porosity. Moreover, the porosity was significantly decreased in the G-TCP5 scaffold (*there were no significant differences between G-TCP0 and G-TCP5 and between G-TCP1 and G-TCP3; † The porosity of G-TCP1 and G-TCP3 was significantly increased compared with that of G-TCP0 and G-TCP5.

The release profile and antibacterial properties

Figure 3 shows the release profiles of vancomycin-loaded composite scaffolds. The G-TCP0 scaffold exhibited the longest duration of vancomycin release with its release duration reaching 56 days. With the increased content of β-TCP granules, the release duration was significantly shortened. Compared with the G-TCP5 scaffold, the composite scaffolds of G-TCP1 and G-TCP3 revealed a slower release of the drug, with the total amount of the drug being released within 42 days. However, the complete release of vancomycin was achieved from the G-TCP5 composite scaffold within 21 days.

Fig. 3

The release profiles of vancomycin-loaded composite scaffolds with different amounts of β-tricalcium phosphate granules (*Significant differences among the four groups, p < 0.05, non-parametric t-test).

Table I shows the inhibition zones of tissue homogenates in different batches of vancomycin-loaded composite scaffolds at different timepoints after implantation. The released vancomycin from the composite scaffolds was bioactive. No effective inhibition zones formed for the tissue homogenate of G-TCP5 after four weeks of implantation.

Biocompatibility

Two weeks after implantation, the gross view of the implanted scaffolds in each group showed that the scaffolds maintained their structures, and they were surrounded by thin fibrous capsules containing vascular vessels without signs of inflammation, such as oedema. Figure 4 shows representative H&E-stained histological sections of vancomycin-loaded composite scaffolds harvested after two weeks of implantation. At this stage, the composite scaffolds maintained their intact interconnecting porous structures. The G-TCP0 scaffold showed no invading host cells inside the implants and only macrophages and host cells were found around the implants. In contrast, the G-TCP1, G-TCP3 and G-TCP5 scaffolds showed prominent infiltration and ingrowth of macrophages and host cells. Figure 4 offers typical histological sections after four weeks of implantation. G-TCP0 and G-TCP1 scaffolds maintained their characteristic microstructural morphology and displayed open pore structures. However, the scaffold integrity was worse compared with those after two weeks of implantation, especially for the G-TCP3 and G-TCP5 scaffolds. After eight weeks of implantation (Fig. 4), the G-TCP0 and G-TCP1 scaffolds still maintained their structures. Only small traces of the G-TCP3 scaffold could be seen, however, the G-TCP5 scaffold could not be found. The histological staining (Fig. 4) showed that all of the composite scaffolds had disintegrated with varying degrees of degradation. The G-TCP5 scaffold was almost completely degraded and the cellular infiltration was clearly decreased. A small number of residual scaffold could be seen in the G-TCP3 scaffold, and the residual scaffold were surrounded by giant cells. The G-TCP0 and G-TCP1 scaffolds were partly degraded, and the host cells and several macrophages were observed within the pores of the composite scaffolds.

Fig. 4

Haematoxylin and eosin-stained histological sections of vancomycin-loaded composite scaffolds harvested at two weeks, four weeks and eight weeks after implantation. Left to right: G-TCP 0, 1, 3, and 5, respectively.

Osteomyelitis model

No animals died during the experiment. Local below-knee swelling and erythema were observed in all animals under physical examination at five days to seven days after the modelling operation. After ten days to 14 days, the wound formed fistulas and had discharge. Three weeks after the osteomyelitis induction, the right hind leg of all of the rabbits showed the typical radiological evidence of chronic osteomyelitis with the following findings: reduced bone density; dead bone; subperiosteal abscess; and formation of new periosteal bone (Fig. 5a). We confirmed that an animal model of chronic osteomyelitis was successfully established as described by Norden.²⁴

Fig.

a) Typical radiological evidence of chronic osteomyelitis: reduced bone density; dead bone; subperiosteal abscesses; and new periosteal bone formation (white arrow: sequestrum formation). b) The haematoxylin and eosin staining showed a large number of clearly visible inflammatory cells, interstitial haemorrhage and bone sequestrum formation (white arrow).

Three rabbits were randomly selected and sacrificed to evaluate the presence of osteomyelitis by pathological H&E staining. Figure 5b presents many visible inflammatory cells, interstitial haemorrhage and bone sequestrum formation.

Treatment of osteomyelitis defects in rabbits

No animal died during the treatment. At different postoperative timepoints after the debridement and implantation of vancomycin-loaded composite scaffolds, radiographic and histological examination were used to evaluate the presence of bone lesion changes, degradation and absorption of the implant material and new bone formation.

The radiological results of animals in the control group showed mild osteolysis after four weeks post debridement, and it became more obvious after eight weeks post debridement, showing increases in osteolytic lesions, development of periosteal reactions, new bone formation and sequestral bone formation. In the G-TCP0 group, radiological signs of infection were nearly absent four weeks after debridement and infections were cured after eight weeks. However, no bone formation was observed in the defect sites at any timepoint. In the G-TCP1 and G-TCP3 groups, no radiological signs of osteomyelitis were observed and the shape of the proximal tibia was restored to its normal structure after four weeks and eight weeks post debridement and scaffold implantation. In the G-TCP1 group, no bone formed after four weeks and the defects were partially repaired after eight weeks. However, in the G-TCP3 group, some callus formed during the first four weeks and the defects were fully repaired at eight weeks post implantation (Fig. 6).

Fig. 6

The radiographic results of animals in the (left to right) control, G-TCP0, G-TCP1 and G-TCP3 groups at four weeks and eight weeks after implantation. top) Four weeks after implantation; bottom) eight weeks after implantation.

Quantitative analysis of the radiographs in Table II revealed that there were no significant differences among the radiological scores of the four groups before scaffold implantation, while significant differences were observed after four weeks and eight weeks post implantation. The radiological scores were gradually decreased after surgery for G-TCP0, G-TCP1 and G-TCP3 groups. The control group had significantly higher mean scores compared with the other groups (p < 0.05, paired t-test). The G-TCP3 group had the best radiological scores after implantation among the four groups. At eight weeks after implantation, the mean scores for the control, G-TCP0, G-TCP1 and G-TCP3 groups were 5.78 (sd 0.13), 2.95 (sd 0.11), 2.46 (sd 0.06) and 0.79 (sd 0.08), respectively. These scores were significantly different (p < 0.05, paired t-test).

Haematoxylin and eosin histological analysis showed that many inflammatory cells were observed, accompanied by the emergence of tissue necrosis, bone structure destruction and sequestrum formation in the control group after four weeks and eight weeks post debridement. In the G-TCP0 group, inflammatory cells were not observed and a small amount of implant material was degraded and absorbed after four weeks post implantation. After eight weeks post implantation, a large amount of material was clearly visible and no new bone had formed. After four weeks post implantation, no signs of inflammation were found in the G-TCP1 group, some materials were degraded and no new bone had formed. However, after eight weeks post implantation, only a small amount of material was visible and some islands of new trabecular bone had formed. In the G-TCP3 group, no evidence of infection was found after four weeks post implantation, a large amount of material was degraded and some new bone had formed. No material was visible after eight weeks post implantation and a large amount of new bone had formed (Fig. 7). The histopathological scores in the G-TCP0, G-TCP1 and G-TCP3 groups decreased with time and these groups could be ranked in a descending order based on histopathologic severity as follows: G-TCP0 > G-TCP1 > G-TCP3 (p < 0.05, paired t-test), showing that the best implantation effect was observed in the G-TCP3 group. The histopathological score in the control group was increased with time, and was significantly higher than those of the other groups at each timepoint (p < 0.05, paired t-test) (Table II).

Fig. 7

Histological observation for the repair of osteomyelitis defects in different groups at eight weeks after implantation. (Hematoxylin-eosin stain × 100) Control group (top left): many inflammatory cells were observed, accompanied by sequestrum formation. G-TCP0 group (top right): most of the composite scaffold was clearly visible and no new bone had formed. G-TCP1 group (bottom left): only small amount of material was visible and some islands of new trabecular bone had formed. G-TCP3 group (bottom right): the composite scaffold was completely degraded and a large amount of new bone had formed.