A MSCs-laden polycaprolactone/collagen scaffold for bone tissue regeneration

Abstract

A mesenchymal stem cell (MSC)-laden scaffold was designed for use in mastoid obliteration. The scaffold consisted of polycaprolactone (PCL) micro/nanofibres, collagen, and cell-laden alginate struts, and was fabricated using a centrifugal melt-spinning, dip-coating, and bioprinting process. As a control, cell-free scaffolds containing the same material composition were used. Both scaffolds were assessed for their abilities to promote osteogenic activities after mastoid obliteration. In vivo mastoid obliteration was performed using guinea pigs that were divided into two groups: those for which the cell-laden scaffold was used and those for which the control scaffold was used. The results revealed that the cell-laden scaffold provided more rapid and broader osteogenesis than the control scaffold. Images obtained by micro-computed tomography and fluorescence microscopy showed that the acceleration of early new bone formation in the cell-laden scaffold was due to the release of osteogenic growth factors and stimulation of the migration and differentiation of host osteoprogenitors caused by MSCs.

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1. Introduction

Surgery for chronic otitis media (COM) aims to create a safe and dry ear with hearing preservation and reconstruction. A canal wall-down (CWD) mastoidectomy is one such common surgical technique with variable long-term outcomes. The potential disadvantages include cosmetic concerns due to an enlarged meatus, complications upon water exposure, and the need for regular maintenance of the mastoid bowl. The CWD mastoidectomy combined with mastoid obliteration results in the formation of a safe, trouble-free cavity.¹ Various materials, both biological and alloplastic, have been used for mastoid obliteration.^2–8

Tissue engineering is a technique for imitating natural tissues, which consist of three components: the extracellular matrix, biomolecules, and cells. We have previously reported on the use of tissue-engineering approaches for mastoid obliterations.^9–11 The incorporation of MSCs into tissue-engineered biomaterials is a popular strategy for accelerating bone formation and osteointegration during bone repair and regeneration.¹² It has been reported that the MSCs can increase the bone forming capacity of the biomaterial in vivo.¹³ In particular, the MSCs increase the number of mesenchymal precursor cells responsible for osteogenic differentiation and bone formation,¹⁴ and these cells also release osteogenic factors to enhance the osteoinductivity of the biomaterial.¹⁵ Moreover, MSCs stimulate hyperbaric microenvironment for new bone formation.¹⁶

Recently, three-dimensional (3D) plotting has been employed by extruding a viscous liquid material (generally a solution, paste, or dispersion) from a pressurised syringe into a liquid medium with matching density. The material is deposited as a long, continuous strut or individual dots from a nozzle or syringe to create 3D ceramics, polymers, or hydrogels.¹⁷ Cell-embedding methods, including cell-printing^18–20 and laser-supplemented photosensitive methods²¹ have also been investigated to create cell-laden scaffolds. These techniques provide homogeneous cell distributions within cultured scaffolds and manipulate the injection of various cell types on a specific region of the scaffold. Although bioprinting methods facilitate the fabrication of living constructs with custom-made architectures using the spatially controlled deposition of multiple bioinks, the low mechanical properties of the cell-printed hydrogel scaffolds have been problematic.²⁰

In the present study, we fabricated a cell-laden scaffold consisting of polycaprolactone (PCL) fibres that were obtained using centrifugal melt-spinning, collagen, and a mixture of alginate and bone marrow-derived mesenchymal stem cells (MSCs) for mastoid obliteration. In the scaffold, the fibrous PCL, collagen, and alginate components were used for mechanical reinforcement. Because there have been no reports on mastoid obliteration using a cell-laden scaffold with MSCs, we evaluated the in vivo effect of a bioprinted PCL/collagen/alginate/MSC scaffold on bone regeneration.

2. Materials and methods

2.1. Fabrication of the scaffold

To fabricate the cell-laden scaffold consisting of the PCL micro/nanofibres, collagen, and MSC-laden alginate, we used PCL (M_w = 45 000 Da, T_m = 60 °C, Sigma-Aldrich, USA), type-I collagen (density = 1.3 g cm⁻³, Matrixen-PSP; Bioland, Cheonan City, South Korea) from porcine tendon, and low-viscosity, high-G-content LF10/60 alginate (FMC BioPolymer, Drammen, Norway).

Using a melt-centrifugal jetting process (temperature = 200 °C, 30-gauge spinneret, and rotating rpm = 1400), a fibrous PCL structure consisting of micro/nanofibrous PCL was obtained (Fig. 1A). After fabricating the fibrous PCL, it was submerged in 2% (w/v) collagen to improve the bioactivity and low hydrophilic property of the fibres. The collagen-coated PCL was immediately placed in a freeze-dryer (FD, SFDSM06, Samwon, South Korea) at −75 °C for 12 h. Collagen was then cross-linked in the fibrous PCL by immersing it in a 50 mM 1-ethyl-(3-3-dimethylaminopropyl)hydrochloride (EDC – 0.579 g) with N-hydroxysuccinimide (NHS – 0.386 g) solution in 99.9% ethanol for 3 h at room temperature (Fig. 1B). After cross-linking, the fibrous PCL was washed three times in 0.1 M Na₂HPO₄ for 2 h each and rinsed four times in demineralised water for 30 min each. After the freeze-drying process, MSC-laden alginate was printed on the surface (pneumatic pressure = 165 kPa; plotting speed = 10 mm s⁻¹) (Fig. 1C). Thus, the fabricated scaffold had three components: the fibrous PCL mat, collagen, and cell-laden alginate. The MSCs were kindly provided by Prof. Eun Ju Lee (Seoul National University Hospital, Seoul, South Korea). The MSC-laden alginate was obtained by mixing alginate (3.5% w/v) with PBS and 0.5% (w/v) CaCl₂ to increase the viscosity of the solution. The mixing ratio of the alginate and CaCl₂ solution was 7 : 3. After mixing, MSCs (density: 1 × 10⁷ mL⁻¹) were incorporated into the alginate solution. After printing the cell-laden alginate on the scaffold, the solution was cross-linked with the 2% CaCl₂ solution prepared in PBS.

Fig. 1 Schematic diagram of the fabrication procedure for the cell-laden scaffold. (A) Centrifugal melt-spinning for polycaprolactone (PCL) fibres. (B) Collagen-coating process. (C) Cell-printing process on the collagen-coated PCL fibrous mat.

2.2. Mechanical test

To measure the mechanical properties, the samples were cut into small strips (10 × 30 mm). Uniaxial tests were performed using a tensile testing machine (Top-tech 2000, Chemilab, South Korea). The stress–strain curves for the scaffolds were recorded at a stretching speed of 0.5 mm s⁻¹. All values are expressed as means ± standard deviation (SD) (n = 5).

2.3. Live/dead analysis

After fabricating the scaffold, it was exposed to 0.15 mM calcein AM and 2 mM ethidium homodimer-1 for 45 min; stained samples were analysed by fluorescence microscope (CKX41; Olympus, Japan). For stained images, green and red indicated live and dead cells, respectively. The ratio of the number of live cells to the total number of cells (including live and dead cells) was calculated using Image-J software, and the value was normalised relative to the initial cell viability (the value before cell-alginate extrusion), as determined using trypan blue (Mediatech, Herndon, VA, USA).

2.4. In vivo study

2.4.1. Surgery. All animal experiments were performed in accordance with the local ethical committee at Chonnam University. Sixteen outbred guinea pigs (male, albino, Samtaco BioKorea, Korea), with normal eardrums and Preyer's reflex, were used for the experiment and were housed separately in sterile cages. Animals were divided into experimental and control groups. Guinea pigs were anesthetised with an intramuscular injection of Zoletil and xylazine. Lidocaine (1%) with 1/100 000 epinephrine was injected into the soft tissue over the tympanic bulla and then a retroauricular incision was made. The bulla was exposed and a hole was created by drilling. After removing the mucoperiosteum of the bulla using a microelevator with alligator forceps, mastoid obliteration was performed using the MSC-laden PCL/collagen/alginate scaffold for the experimental group I (n = 7), and the PCL/collagen/alginate scaffold for the control group II. The wounds were then closed, and ciprofloxacin was administered by intramuscular injection to prevent infection.

2.4.2. Fluorescent bone labelling. Animals were administered fluorescent bone labels for the evaluation of sequential bone formation. To assess the active mineralisation of new bone, each group received calcein blue at 3 weeks, oxytetracycline hydrochloride at 5 weeks, and alizarin red at 7 weeks (Sigma-Aldrich Chemical Co. St Louis, USA).

2.4.3. Ex vivo micro-computed tomography. Animals were sacrificed 8 weeks after surgery and micro-computed tomography (micro-CT) images were obtained from each bulla using a volumetric CT scanner (SkyScan 1172 high-resolution micro-CT, Kontich, Belgium).

2.4.4. Histological evaluation. After bullae were harvested, they were fixed in formaldehyde solution (10%) for 48 h and embedded in a glycol methacrylate solution, Technovit 7200 VLC (Kultzer & Co, Wehrhein, Germany). Following polymerisation, samples were processed using a sawing and grinding technique. After examination of fluorescent labelling using confocal microscope (Leica, Wetzlar, Germany), the samples were stained with hematoxylin and eosin. A histomorphometric analysis of the middle portion of each group was performed using a PC-based image analysis system (Image Inside, Focus Technology, Daejon, Korea). All values are presented as the mean ± SD. Significance was set at 5% and p-values were adjusted for multiple comparisons.

3. Results

3.1. Fabricated cell-laden scaffold and morphology

Fig. 2A and B show the optical images of the fibrous PCL/collagen/alginate (PCA) and MSC-laden PCL/collagen/alginate (PCAMSC) scaffolds, respectively. As shown in the surface images of the PCA and PCAMSC scaffolds, the printed strut size and pore size on the collagen layer was ∼423 ± 27 μm and ∼521 ± 26 μm, respectively. The height of the printed layer was 950 μm and the cell density of the printed strut was 128 cells mm⁻³. The average diameter and thickness of the fabricated scaffolds were 5 mm and 1.2 mm, respectively. Fig. 3A shows surface and cross-sectional scanning electron microscopy (SEM) images of the centrifugally fabricated PCL fibrous mat. The fibres had a variable diameter distribution due to the stretching motion of the melted fibres from centrifugal and shear forces. Fig. 3B shows the distribution of fibre diameters for centrifugally spun micro/nanofibres. The average diameter of the fibres was 7.7 ± 6.5 μm. Collagen fully covered the fibrous mat and slightly infiltrated the PCL fibrous mat (Fig. 3C). Fig. 3D and E show SEM images of the PCA and PCAMSC surfaces printed with pure alginate (control) and a mixture of alginate/MSCs, respectively. As shown in the magnified SEM image of Fig. 3E, the MSCs were incorporated into the PCAMSC scaffold. In Fig. 3F, cross-sectional optical, SEM, and fluorescence (live cells) images of the alginate/cell-coated PCAMSC are provided. As shown in the images, the MSCs are not only on the surface, but also within the alginate struts. To show the release behavior of the MSCs at 1 and 10 days, optical images of the released cells were illustrated in Fig. 3G.

Fig. 2 Optical images of (A) PCL/collagen/alginate (PCA) and (B) PCL/collagen/alginate mesenchymal stem cell (PCAMSC) scaffolds.

Fig. 3 (A) Surface and cross-sectional scanning electron microscopy (SEM) images of the PCL fibrous mat, and (B) the distribution of the fibre diameter. (C) Surface and cross-sectional SEM images of the collagen-coated fibrous mat. SEM images of (D) the alginate-coated surface (PCA) and (E) the alginate/cell-coated surface (PCAMSC) on the collagen/PCL fibrous mat. (F) Cross-sectional optical, SEM, and fluorescence (live cells) images of the alginate/cell-coated PCAMSC. (G) Optical images of released cells at 1 and 10 days of cell culture.

3.2. Mechanical stress test

For regenerating bone tissues, biomedical scaffolds should have sufficient mechanical strength to withstand new tissue formation. To evaluate the mechanical properties of the scaffolds, a tensile test was performed. Fig. 4 shows the stress–strain curves for the pure fibrous PCL mat, collagen-coated scaffold, and PCAMSC scaffold. The elastic modulus of each material and the modulus of the pure PCL fibrous mat, collagen-coated PCL mat, and PCAMSC was 4.6 ± 0.2 MPa, 5.1 ± 0.5 MPa, and 8.4 ± 1.6 MPa, respectively.

Fig. 4 Tensile properties for pure PCL, a collagen-coated PCL mat (PC), and PCAMSC scaffolds.

3.3. Live and dead cells in the scaffold

Fig. 5A and B show the combined fluorescence/optical images of the PCA and PCAMSC scaffolds, respectively, after cell-printing for 4 h. The green and red indicate live and dead cells, respectively. As expected for the PCA scaffold, live and dead cells were not observed, while for the PCAMSC scaffold, well-distributed live cells were observed. Using the number of live and dead cells, the cell viability of the PCAMSC scaffold was calculated using Image-J software. The initial cell viability of the cell-laden PCAMSC scaffold was 93.4 ± 1.1%.

Fig. 5 Fluorescence images (green = live; red = dead) of (A) PCA and (B) PCAMSC scaffolds.

3.4. Micro-CT findings

No adverse events were observed during the healing interval. The CTs of bulla cavities 8 weeks after obliteration showed the state of bone regeneration. Diffuse radio-opaque regions were clearly visible at each bulla in the experimental group, while an inadequate marginal radio-opaque line was observed in the control group. Limited bone formation was observed in the control group (Fig. 6).

Fig. 6 Micro computed tomography (micro-CT) images show the more prominent new bone formation in the cell-laden scaffold (PCAMSC), compared with the control scaffold (PCA). Arrows indicate osteogenesis.

3.5. Light microscope images

Histological observations showed that a significant amount of new bone was formed in the experimental group where both mature and immature osteogenesis was visible. In contrast to the experimental group, limited osteogenesis was observed in the control group (Fig. 7A). Histomorphometric analyses showed significant differences between the two groups (control vs. experimental, p = 0.247) (Fig. 7B).

Fig. 7 (A) Diffuse new-bone formation in the PCAMSC group, compared with the PCA group. The empty space in the mastoid cavity is prominent in the PCA group. (B) The histomorphometry data revealed significant differences between the two groups (p = 0.0247). These data are in agreement with the histological findings.

3.6. Confocal microscopic findings

Confocal microscopic findings revealed that three distinct colours corresponding to sequential osteogenesis were observed in the experimental group, whereas poor sequential osteogenesis was observed in the control group (Fig. 8).

Fig. 8 Confocal microscope images showed enhanced osteogenesis in the PCAMSC group, compared with the PCA group. Blue, calcein; green, oxytetracycline; and red, alizarin.

4. Discussion

Multiple studies have reported the shortcomings, limitations, and complications associated with the current clinical treatments for mastoid obliteration. These treatments include autologous and allogeneic transplantations using autografts and allografts.^1–8 To date, autografts are the gold standard for bone grafts because they are histocompatible, non-immunogenic, and they provide all of the properties of bone graft materials. Typically a bone pate is used for mastoid obliteration; however, because bone pates can be contaminated by infected mucosa or squamous epithelium, it should be obtained from the lateral cortical bone.²²

Allogeneic bone is also histocompatible and available in various forms, including as a demineralised bone matrix (DBM), and morcellised or cancellous chips, depending on the host-site requirements. Compared with autografts, allografts are associated with the increased risk of immunoreactions and transmission of infections.

To date, the development of bone tissue engineering scaffolds has been for bone regeneration. For mastoid obliteration, we have reported effective osteogenesis using a tissue-engineered bone scaffold. Over the past two years, remarkable progress has been made in the development of tissue engineering techniques and strategies. The therapeutic roles of MSCs have been studied due to their tissue-repair activity,²³ which has been attributed to a paracrine effect due to the production of several factors. Additionally, MSCs induce the host-cell production of soluble molecules, such as growth factors.²⁴ MSC therapy has been shown to promote bone regeneration by increasing the formation of new bone and increasing bone mineral density.²⁵ Stratified analyses showed that new bone formation was enhanced when a larger number of cells were present and a scaffold was used, compared with direct cell injection methods.²⁴

In this study, we fabricated a cell-laden PCAMSC scaffold. The centrifugally obtained PCL fibres varied in diameter; however, solvent-free PCL fibres are advantageous as biomedical scaffolds, compared with solvent-based electrospun micro/nanofibres. Our previous work showed that osteogenic gene expression of BMP-2 and OCN from pre-osteoblasts cultured on the melt-spun poly(lactic acid) fibres were 6-fold and 1.8-fold higher than that of the solvent-based electrospun fibres. Such changes in gene expression were specifically due to solvent-free conditions.²²

For the cell-laden PCAMSC scaffold, the initial cell viability after printing was approximately 93.4 ± 1.1%. These data suggest that the cell-printing process is suitable for the fabrication cell-laden scaffolds. The tensile modulus of the cell-laden scaffold (PCAMSC) was also higher than that of the pure PCL mat and collagen-coated PCL mat, because alginate cross-linking contributes to the stiffness and strength of the scaffold. Moreover, the mechanical properties of the cell-laden structure were significantly enhanced compared with pure cell-laden alginate.

Using the cell-laden PCAMSC scaffold, bone regeneration was observed within 8 weeks, with osteogenesis occurring earlier in the experimental group, compared with the control group. The bio-printed scaffold was soft and malleable, and easily implanted into defective areas. The incorporation of MSCs into tissue-engineered biomaterials is a widely studied strategy for accelerating bone formation and osteointegration during bone repair and regeneration.²⁵ The mechanisms by which enhanced bone regeneration occurs involves directly providing MSCs that contribute to osteogenic differentiation and bone formation, as well as enhanced osteoinductivity of the biomaterial via the release of osteogenic growth factors and stimulation of the migration and differentiation of host osteoprogenitors.^26–28

In the present study, bone regeneration was more effective in the experimental group, compared with the control group. The findings from the micro-CT images agreed with the histological findings, and the three fluorescent stains used to assess bone formation indicated that osteoinductivity was enhanced in the experimental group containing MSCs, compared with the control group lacking MSCs.

We previously showed that a 3D PCL scaffold coated with bone morphogenic protein (BMP)-2 or umbilical cord serum^9,10 enhanced osteogenesis. In this study, we developed a PCAMSC scaffold, lacking a BMP-2 coating, which also enhanced early new bone formation. Previous studies showed that rhBMP-2 induces an inflammatory reaction that results in massive edema and subsequent axillary vein compression,²⁹ and inhibits bone regeneration.³⁰ Lacey et al.³⁰ reported that osteogenic differentiation from the MSC is suppressed by interleukin-1 beta and tumour necrosis factor alpha. Bone mineralisation was also suppressed by these factors.

In the present study, the PCAMSC scaffold increased bone regeneration after mastoid obliteration, and the use of the PCAMSC scaffold yielded superior bone regeneration, compared with direct MSC transplants.

5. Conclusion

In this study, an innovative cell-laden scaffold (PCAMSC) consisting of centrifugally obtained micro/nanofibrous PCL mat, collagen, and cell (MSC)-mixed alginate was developed. The mechanical properties of the PCAMSC scaffold were superior to that of pure centrifugally obtained PCL fibres, and the survival of MSCs in the scaffold was adequate. These in vivo data suggest that the MSC-laden cell-printed PCAMSC scaffolds were more effective at promoting bone formation, compared with a simple PCA scaffold.

Acknowledgements

This research was financially supported by a Grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. A120942).

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Footnote

† The authors equally contributed in the work.

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