Construction of mesenchymal stem cell-containing collagen gel with a macrochanneled polycaprolactone scaffold and the flow perfusion culturing for bone tissue engineering

Abstract

A novel bone tissue-engineering construct was developed by using poly(ɛ-caprolactone) (PCL)-macrochanneled scaffolds combined with stem cell-seeded collagen hydrogels and then applying flow perfusion culture. Rat mesenchymal stem cells (MSCs) were loaded into collagen hydrogels, which were then combined with macrochanneled PCL scaffolds. Collagen hydrogels were demonstrated to provide favorable growth environments for MSCs and to foster proliferation. Cell number determination identified retention of substantially fewer (50-60%) cells when they were seeded directly onto macrochanneled PCL than of cells engineered within collagen hydrogels. Additionally, the cells actively proliferated within the combined scaffold for up to 7 days. MSC-loaded collagen-PCL scaffolds were subsequently cultured under flow perfusion to promote proliferation and osteogenic differentiation. Cells proliferated to levels significantly higher in flow perfusion culture than that under static conditions during 21 days. A quantitative polymerase chain reaction (QPCR) assay revealed significant alterations in the transcription of bone-related genes such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP), such as 8-, 2.5-, and 3-fold induction, respectively, after 10 days of flow perfusion relative to those in static culture. OPN and OCN protein levels, as determined by Western blot, increased under flow perfusion. Cellular mineralization was significantly enhanced by the flow perfusion during 21 and 28 days. Analyses of mechanosensitive gene expression induced by flow perfusion shear stress revealed significant upregulation of c-fos and cyclooxygenase-2 (COX-2) during the initial culture period (3-5 days), suggesting that osteogenic stimulation was possible as a result of mechanical force-driven transduction. These results provide valuable information for the design of a new bone tissue-engineering system by combining stem cell-loaded collagen hydrogels with macrochanneled scaffolds in flow perfusion culture.

Keywords: 3D scaffolds; bone tissue engineering; collagen gel; flow perfusion; osteogenic differentiation.

FIG. 1.

Schematic illustration of the experimental design of the study: (a) cell-scaffold design and (b) flow perfusion system. MSCs were initially pooled in collagen solution, which was subsequently applied to the macrochannels of PCL scaffolds to form cell–collagen-embedded scaffold constructs (a). The constructs were cultured using a flow perfusion system supplied with osteogenic culture medium (b). The constructs were placed into a chamber contained in an incubator with a humidified atmosphere of 5% CO₂ at 37°C. Fresh medium was supplied continuously at a constant rate (0.6 mL/min) using a peristaltic pump, and the exhausted medium was collected and discarded. MSCs, mesenchymal stem cells; PCL, poly(ɛ-caprolactone).

FIG. 2.

Scanning electron microscopy morphology of (a, b) macrochanneled PCL scaffold produced by robotic dispensing and (c, d) collagen-treated PCL scaffold at different magnifications. The PCL scaffold showed a well-defined pore configuration with pore size of 225 μm (±21 μm) and framework diameter of 213 μm (±16 μm). Square-shaped pores were developed to form channels penetrating through the scaffold (a). The surface of the scaffold was smooth, as is characteristic of a solidified PCL polymer (b). A layer-by-layer deposition process resulted in 10 consecutive layers. The scaffold was constructed with a dimension of 10 mm×10 mm×∼3 mm for cell culture. PCL scaffolds were soaked in collagen solution and then freeze-dried to show the appearance of dried collagen mostly filling the pore channels (c) and the fibrous morphology of collagen on the surface (d). Scale bar=100 μm.

FIG. 3.

Comparison of initial cell loading (at 24 h) and extended proliferation (for up to 7 days) between cells directly seeded onto PCL scaffolds and those pooled in collagen gel combined with PCL scaffolds. More than 50% of cells easily penetrated through scaffold macrochannels to sediment in the bottom of the culture dish, resulting in a reduction in the number of adherent cells when seeded directly. In contrast, cells contained within collagen gels were predominantly retained on the scaffolds. The MTS assay showed that cell viability almost doubled with the use of collagen gels (data at 24 h; **p<0.01, n=5). Cells continued to proliferate for up to 7 days in both cases; however, significant differences in initial cell loading were maintained during the proliferative stage (data at 3 and 7 days; **p<0.01, n=5), demonstrating that high initial cell loading is important for securing a large cell population during prolonged culture. MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

FIG. 4.

Effects of flow perfusion culture on the proliferation of MSCs contained in collagen/PCL scaffolds over culture periods of up to 21 days. Twenty-four hours after applying the MSC–collagen gel (set as t=0), cell–scaffold constructs were cultured either under a normal static condition with the medium refreshed every 2 days or under flow perfusion at a constant rate of ∼0.6 mL/min using a peristaltic pump. Osteogenic medium containing ascorbic acid, dexamethasone, and β-glycerophosphate was used to induce osteogenesis of MSCs. After culture for 14 and 21 days, the MTS assay was conducted to measure cell proliferation; data are represented with respect to the onset of osteogenic culture (24 h after MSC–collagen gel treatment, which was set as t=0). The flow perfusion condition resulted in significantly higher cell proliferation at all culture periods compared to that of static culture (**p<0.01 at day 14 and *p<0.05 at day 21, n=5).

FIG. 5.

CLSM cell morphology during static (a, c, e) and flow perfusion (b, d, f) culture at different time points: (a, b) 3, (c, d) 14, and (e, f) 21 days. Perfusion culture significantly enhanced cell proliferation, particularly for prolonged periods. While the cells loaded in concert with the collagen gel were distributed within the macrochannels of the PCL scaffold at the initial culture period, prolonged culture allowed cells to move toward the surface of the PCL scaffold, which was more pronounced by perfusion culture. Scale bar=100 μm. CLSM, confocal laser-scanning microscopy.

FIG. 6.

ALP activity, an early osteogenic marker, in MSCs during the culture of combined scaffold–MSC constructs under a static or perfusion condition. ALP enzymatic activity was normalized to total protein content, and results are represented with respect to that of static culture at 7 days. ALP levels increased with culture time under both culture methods; the increase was greater under flow perfusion system, resulting in significantly higher enzymatic activity at 14 days in flow perfusion culture than that under the static condition (**p<0.01, n=5). ALP, alkaline phosphatase.

FIG. 7.

MSC expression of genes associated with osteoblastic differentiation as analyzed by QPCR. Gene expression levels are presented with respect to those in static culture at each culture time. The mRNA levels of essential osteogenic genes, including (a) OPN, (b) OCN, and (c) BSP, were significantly upregulated by flow perfusion culture at 10 days (**p<0.01 for all genes, n=3), although no significant difference was noted at 5 days. Induction was as high as 8-, 2.5-, and 3.0-fold for OPN, OCN, and BSP, respectively. QPCR, quantitative polymerase chain reaction; OPN, osteopontin; OCN, osteocalcin; BSP, bone sialoprotein.

FIG. 8.

CLSM immunofluorescent images of cells cultured under (a, c, e) static and (b, d, f) perfusion conditions showing green fluorescent staining of OPN (a, b), OCN (c, d), and BSP (e, f) expression after 10 days of culture. Fluorescence was qualitatively more pronounced in cells cultured under flow perfusion than those under a static culture condition.

FIG. 9.

Western blot analysis of OPN and OCN proteins during culture for 20 days: (a) protein expression bands (OPN at 66 kDa and OCN at 37 kDa, and glyceraldehyde 3-phosphate dehydrogenase used as a reference) and (b) quantification of band intensity by image densitometry. Of special note is the more distinct expression of both protein bands (especially OPN) under flow perfusion than that under static culture. Image densitometry analysis from two sets of experiments showed significantly higher expression levels under flow perfusion than those under static culture (**p<0.01, n=2).

FIG. 10.

Mineralization assay of the cells cultured under either static or perfusion conditions for 21 and 28 days. Ca content was determined quantitatively using a Calcium E-Test kit. The mineralization level was significantly higher in perfusion culture than in static at day 28 (**p<0.01, n=3).

FIG. 11.

Quantitative analysis of c-fos and COX-2 gene expression by QPCR; these genes are related to osteogenic differentiation downstream of mechanotransduction. Combined scaffold–MSC constructs were cultured for relatively short periods (1, 3, or 5 days) under static or flow perfusion conditions. QPCR analyses showed significantly enhanced induction of both genes after 3 and 5 days in flow perfusion culture compared to that under a static condition (**p<0.01, n=3). COX-2, cyclooxygenase-2.