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Original article
Three-dimensional polycaprolactone– hydroxyapatite scaffolds combined with bone marrow cells for cartilage tissue engineering
Journal of Biomaterials Applications 0(0) 1–11 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215575762 jba.sagepub.com
Bo Wei1,2,3, Qingqiang Yao1,2,3, Yang Guo1,2,3, Fengyong Mao1,2, Shuai Liu1,2, Yan Xu1,2,3 and Liming Wang1,2,3
Abstract The goal of this study was to investigate the chondrogenic potential of three-dimensional polycaprolactone–hydroxyapatite (PCL–HA) scaffolds loaded with bone marrow cells in vitro and the effect of PCL–HA scaffolds on osteochondral repair in vivo. Here, bone marrow was added to the prepared PCL–HA scaffolds and cultured in chondrogenic medium for 10 weeks. Osteochondral defects were created in the trochlear groove of 29 knees in 17 New Zealand white rabbits, which were then divided into four groups that underwent: implantation of PCL–HA scaffolds (left knee, n ¼ 17; Group 1), microfracture (right knee, n ¼ 6; Group 2), autologous osteochondral transplantation (right knee, n ¼ 6; Group 3), and no treatment (right knee, n ¼ 5; Control). Extracellular matrix produced by bone marrow cells covered the surface and filled the pores of PCL–HA scaffolds after 10 weeks in culture. Moreover, many cell-laden cartilage lacunae were observed, and cartilage matrix was concentrated in the PCL–HA scaffolds. After a 12-week repair period, Group 1 showed excellent vertical and lateral integration with host bone, but incomplete cartilage regeneration and matrix accumulation. An uneven surface of regenerated cartilage and reduced distribution of cartilage matrix were observed in Group 2. In addition, abnormal bone growth and unstable integration between repaired and host tissues were detected. For Group 3, the integration between transplanted and host cartilage was interrupted. Our findings indicate that the PCL–HA scaffolds loaded with bone marrow cells improved chondrogenesis in vitro and implantation of PCL–HA scaffolds for osteochondral repairenhanced integration with host bone. However, cartilage regeneration remained unsatisfactory. The addition of trophic factors or the use of precultured cell–PCL–HA constructs for accelerated osteochondral repair requires further investigation. Keywords Polycaprolactone, hydroxyapatite, bone marrow cells, cartilage tissue engineering, three dimensional
Introduction Healthy articular cartilage has low vascularity, which endows it with limited ability to respond to injury. The primary aim of cartilage repair is to restore the structure and function of damaged cartilage.1,2 Many surgical interventions are widely available for treating damaged cartilage.3–7 Among them, microfracture is highly appreciated because of its minimal invasiveness, simplicity, and low cost.3,8,9 However, the repaired tissues are composed mainly of fibrocartilage, which has inferior biomechanical properties compared to hyaline cartilage.10 Eventually, discontinuous lateral integration, abnormal bone growth, and degenerative cartilage
1 Department of Orthopaedic Surgery, Nanjing First Hospital, Nanjing Medical University, Nanjing, China 2 Cartilage Regeneration Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China 3 China-Korea United Cell Therapy Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
Corresponding author: Liming Wang, Department of Orthopedic Surgery, Nanjing First Hospital, Nanjing Medical University, PO Box 210006, Nanjing 210006, Jiangsu, People’s Republic of China. Email: [email protected]
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develop.11,12 Moreover, the clinical outcomes after microfracture-based cartilage repair of the knee are age dependent, and patients aged 40 years or younger with lesions on the femoral condyles experience the best outcomes.13 Additionally, autologous osteochondral transplantation (AOT) has been widely used because it can achieve fine vertical fixation, followed by bone healing and structural reconstitution.4,14 However, the lateral integration between the osteochondral plugs and the surrounding host cartilage is limited, which can lead to poor mechanical properties at the interface regions and eventually cause cartilage degeneration.15,16 Therefore, various biomaterial scaffolds have been investigated to improve cartilage repair. Generally, an ideal scaffold for cartilage repair should demonstrate mechanical properties that resemble those of native cartilage, promote the concentration of cartilage-like extracellular matrix (ECM), and integrate well with the surrounding host tissues.2 Moreover, scaffolds fabricated for cartilage tissue engineering usually possess a porous structure that facilitates cell adhesion, proliferation, and differentiation.17–19 Previously, we developed a porous bone marrow mesenchymal stem cell– derived (BMSC–d) ECM scaffold and investigated the chondrogenesis within the BMSC–dECM scaffold loaded with bone marrow cells.20,21 However, the mechanical properties of the BMSC–dECM scaffold are inferior to those of native cartilage, suggesting that the scaffolds cannot withstand long-term load bearing during the repair of articular cartilage. Recently, rapid prototyping (RP) techniques have been developed for fabricating scaffolds with controllable microstructures and properties.22,23 Thus, scaffolds with controlled porosity and mechanical strength can be obtained using RP techniques. Polycaprolactone (PCL) is a nontoxic, slow degrading, and tissue compatible polymer that can be used as a biomaterial for tissue engineering applications.24,25 Hydroxyapatite (HA), which demonstrates chemical (a)
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constituents resembling the mineral components of bone, is widely considered suitable for bone tissue engineering.26,27 Furthermore, many groups have investigated blends of PCL and HA as scaffolds offering improved mechanical properties, cell adhesion and proliferation, and osteoconductivity.28–30 However, rare studies have examined the chondrogenic potential of a porous PCL–HA scaffold with controllable pore size, fine biocompatibility, and mechanical properties critical for cartilage tissue engineering.31,32 Thus, in the present study, we tested our hypothesis that a three-dimensional PCL–HA scaffold loaded with bone marrow cells can enhance cartilage regeneration in vitro and improve osteochondral repair in vivo.
Materials and methods Fabrication of three-dimensional PCL–HA scaffolds with controllable microstructure The three-dimensional properties of PCL–HA scaffolds were designed using computer-aided design software. First, 70 wt% PCL (molecular weight65,000, Sigma Chemical Co., St. Louis, MO, USA) was mixed with 30 wt% HA power (Sigma Chemical Co.) in a slurry at 120%. The PCL–HA scaffolds were fabricated using a three-dimensional layer-by-layer fused deposition modeling (FDM) 3000 system (Stratasys Inc., USA). The obtained PCL–HA scaffolds had a diameter of 6 mm, a thickness of 3 mm, and an average pore size of 400 mm (Figure 1(a, b)). The fabricated PCL–HA scaffolds were sterilized using ethylene oxide for subsequent in vitro chondrogenesis and in vivo implantation experiments.
In vitro chondrogenesis in PCL–HA scaffolds loaded with bone marrow cells All animal experiments were approved by the Institutional Review Board and the Animal Research (c)
Figure 1. Gross and microscopic observation of and cell viability within polycaprolactone/hydroxyapatite (PCL–HA) scaffolds. (a) The porous PCL–HA scaffolds consisting of 70 wt% PCL and 30 wt% HA were fabricated using a rapid prototyping technique. (b) Scanning electron microscopy (SEM) images showed that the porous PCL–HA scaffolds had an average pore size of 400 mm. MagniEcation25. (c) Confocal microscopy images demonstrated most live cells emitted green fluorescence, and few dead cells were labeled with red fluorescence after seven days in culture. Magnification100.
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Wei et al. Committee of Nanjing Medical University. Bone marrow samples were aseptically obtained from the posterior superior iliac spine of 17 female New Zealand white rabbits (aged four to six months, weighting 3.5–4.0 kg) via the bone marrow puncture procedure. Then, the prepared PCL–HA scaffolds were immersed in the collected bone marrow for 40 min at room temperature to ensure good contact between PCL–HA scaffolds and bone marrow cells. The obtained composite scaffolds were immersed in chondrogenic medium (high-glucose Dulbecco’s modified Eagle’s medium [Gibco, Australia], 10% fetal bovine serum [Gibco, Australia], 1% penicillin–streptomycin, 40 mg/ml L-proline, 100 mg/ml sodium pyruvate, 37.5 mg/ml L-ascorbic acid 2-phosphate, 6.25 mg/ml bovine insulin, 6.25 mg/ml transferrin, 6.25 mg/ml selenous acid, 5.33 mg/ml linoleic acid, 100 nM dexamethasone, and 10 ng/ml transforming growth factor-b3 [TGF-b3; PeproTech, Rocky Hill, NJ, USA])21 for 10 weeks in vitro.
Cell viability, gross observation, density, and microstructure Cell viability in the PCL–HA scaffolds was evaluated after seven days in culture using the Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit (Molecular Probes/Invitrogen, Eugene, OR, USA). Briefly, samples were collected and washed with phosphate-buffered saline, incubated in the combined LIVE/ DEAD assay reagents for 30 min at room temperature, and then observed using a confocal microscope (Leica TCS SP2 Confocal Microscope; Leica, Mannheim, Germany). Live cells are marked with green fluorescence, and dead cells are labeled with red fluorescence. The macroscopic images of PCL–HA scaffolds were observed after 10 weeks in vitro. Then, the PCL–HA scaffolds were scanned to detect the calcification of matrix via micro-computed tomography (micro-CT; Hiscan MCT-1108, Nanjing, China) with a scanning width of 50 mm. The surface of PCL–HA scaffolds was positioned vertical to the axis of the ray beam. Meanwhile, a native osteochondral plug was scanned as a control. Additionally, the microstructures of both surface and transverse sections of the PCL–HA scaffolds were evaluated by scanning electron microscopy (SEM; Hitachi S4800, Tokyo, Japan).
Histology and immunohistochemistry After 10 weeks in culture, the harvested PCL–HA scaffolds were fixed in 10% neutral formalin for 24 h, decalcified in 5% nitric acid for 48 h, dehydrated using a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections (5 mm in thickness) were stained
3 with hematoxylin–eosin (H&E) and Safranin-O/FastGreen (Safranin O), separately. For immunohistochemical staining, sections were sequentially exposed to proteinase K for 30 min, 3% hydrogen peroxide in methanol for 10 min, 10% goat serum for 30 min, and mouse antitype II collagen monoclonal antibody (Novus Biologicals, Littleton, CO, USA) for 90 min, followed by goat anti-mouse secondary antibody (Nanjing KeyGen Biotech. Co., Ltd., Jiangsu, China) for 30 min and 3,30 -diaminobenzidine tetrahydrochloride solution for 3–5 min. Then, the sections were counterstained with Mayer’s hematoxylin (Sigma) for 8 min and mounted with neutral balsam.
Biochemical analysis After 1 and 10 weeks in culture, the total DNA content of PCL–HA scaffolds was measured using a Quit-iT dsDNA kit (Molecular Probes/Invitrogen, Eugene, OR, USA) (n ¼ 3). Briefly, the samples were digested with papain and reacted with Hoechst dye 33258 for 30 min. Fluorescence intensity was detected with a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) with excitation at 360 nm and emission at 460 nm. DNA from salmon testes (Sigma) was used to create a standard curve. The glycosaminoglycan (GAG) content of PCL–HA scaffolds was measured using a BlyscanTM sulfated GAG assay kit (Biocolor, Newtonabbey, UK) (n ¼ 3). In brief, a papain-digested solution was mixed with Blyscan dye reagent and centrifuged at 12,000 r/min for 10 min. The nonbound GAG dye was then removed, and dissociation reagent was added. A microplate reader (Bio-Rad Laboratories) was used to measure absorbance at 656 nm. The collagen content of PCL–HA scaffolds was quantified using a Sircol Collagen Assay Kit (Biocolor) (n ¼ 3). Briefly, the samples were digested with pepsin, mixed with Sircol dye reagent, and centrifuged at 12,000 r/min for 10 min. The deposit was then washed with acid–salt wash reagent and dissolved in alkali reagent. A microplate reader (Bio-Rad Laboratories) was used to measure the absorbance at 555 nm.
Surgical treatment and grouping A total of 17 New Zealand white rabbits, which were used for bone marrow collection to assay in vitro chondrogenesis, were anesthetized by intramuscular administration of ketamine hydrochloride (15 mg/kg). Osteochondral defects (6 mm 6 mm 3 mm) were created in the trochlear groove of 29 knees. PCL–HA scaffolds unseeded with cells were implanted into the defects of left knees (n ¼ 17; Group 1). Microfracture holes were created in the defects of right knees (n ¼ 6;
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4 Group 2). Osteochondral plugs, which were harvested from the osteochondral defects, were reimplanted into the defects of right knees (n ¼ 6; Group 3). Meanwhile, untreated knees were designated as controls (n ¼ 5; Control). Twelve hours after implantation, two rabbits (one with the left knee in Group 1 and the right knee in Group 2, and another with the left knee in Group 1 and the right knee in Group 3) were sacrificed using an overdose of sodium pentobarbital to observe the immediate filling. At 12 weeks after implantation, the remaining rabbits were sacrificed for further analysis.
Macroscopic evaluation, histological analysis, and immunohistochemical staining of repaired tissues Knee joints (distal femurs) were harvested, and the gross morphology (including that of both surface and longitudinal sections) of repaired tissues was assessed after 12 weeks of repair in vivo. Then, the samples were fixed in 10% neutral formalin for 24 h, decalcified in 5% nitric acid for 96 h, and embedded in paraffin. Sections (5 mm in thickness) were stained with H&E and Safranin O to evaluate cell morphology, proteoglycan distribution, and integration with surrounding host tissues (including both vertical and lateral integration). Separate sections (5 mm in thickness) were incubated with mouse antitype II collagen monoclonal antibody (Novus Biologicals, Littleton, CO, USA) and mouse antitype I collagen antibody (Sigma–Aldrich, St. Louis, MO, USA) for 90 min at 37 C to observe type II and type I collagen production, respectively.
Statistical analysis All data are expressed as the mean standard deviation. The unpaired Student’s t-test was used to compare the differences in the total DNA content, GAG content, and collagen content of PCL–HA scaffolds between 1 and 10 weeks in culture. The level of statistical significance was set at P < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software (version 13.0; SPSS Inc., Chicago, IL, USA).
Results Cell viability and macroscopic and microscopic observations of PCL–HA scaffolds loaded with bone marrow cells After seven days in culture, bone marrow cell viability in the PCL–HA scaffolds was evaluated using confocal microscopy. Many live cells emitted green fluorescence and were distributed on the fibers and around the pores of PCL–HA scaffolds. Meanwhile, few dead cells
Journal of Biomaterials Applications 0(0) emitting red fluorescence were unevenly distributed in the PCL–HA scaffolds (Figure 1(c)). After 10 weeks in culture, many hyaline-like tissues had formed on the surface of PCL–HA scaffolds, particularly in the peripheral regions of the scaffolds. Moreover, the pores of scaffolds were uniformly filled (Figure 2(a)). Micro-CT images showed that the physical density of PCL–HA scaffolds resembled that of cartilage, but were lower than that of bone (Figure 2(b, c)). Furthermore, SEM images demonstrated that both the surface and transverse sections of PCL–HA scaffolds were evenly covered with ECM, indicating that proliferation and differentiation of marrow cells occurred in the PCL– HA scaffolds (Figure 2(d, e)).
Histological, immunohistochemical, and biochemical analyses of PCL–HA scaffolds loaded with bone marrow cells After 10 weeks in culture, H&E staining of PCL–HA scaffolds showed that many cell-laden cartilage lacunae were distributed throughout the scaffolds (Figure 3(a,d)). Safranin O and type II collagen staining demonstrated that GAG and type II collagen were unevenly concentrated in the fibers of PCL–HA scaffolds (Figure 3(b, c, e, f)). Furthermore, the contents of DNA, GAG, and collagen in PCL–HA scaffolds were detected after 1 and 10 weeks in culture, individually. The average total DNA content at 10 weeks (2.26 0.17 mg/mg) was significantly higher than that at one week (0.96 0.08 mg/mg; P < 0.001; Figure 4(a)). Similarly, the contents of GAG and collagen at 10 weeks (27.19 0.90 mg/mg GAG; 50.77 2.83 mg/mg collagen) were significantly higher than those at one week (5.35 0.23 mg/mg GAG; 8.38 0.68 mg/mg collagen; P < 0.001; Figure 4(b, c)).
Macroscopic evaluation of repaired tissues No obvious effusions or inflammative reactions were detected in the knee joints of all rabbits. After the PCL–HA scaffolds were implanted into the osteochondral defects (Figure 5(a)), Group 1 showed that the scaffolds were firmly fixed in the defects and well contacted by marrow clots by 12 h following microfracture (Figure 5(d)). After microfractures were created in the knee joints (Figure 5(b)), Group 2 demonstrated that osteochondral defects filled with marrow clots after 12 h. Moreover, the repair tissues were integrated well with surrounding tissues (Figure 5(e)). After autologous osteochondral plugs were retransplanted into the osteochondral defects (Figure 5(c)), Group 3 showed that the cartilage surface of plugs was aligned with the surface of the host cartilage after 12 h. However, the integration between osteochondral plugs and host
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Figure 2. Macroscopic and microscopic evaluations of PCL–HA scaffolds loaded with bone marrow cells after 10 weeks in culture. (a) Gross observation demonstrated that hyaline-like tissues had formed over the surface of PCL–HA scaffolds. (b, c) Micro-CT images revealed that the physical density of PCL–HA scaffolds resembled that of cartilage, but was lower than that of bone. (d, e) SEM images showed that both the surface and transverse sections of PCL–HA scaffolds were evenly distributed with extracellular matrix (ECM). Magnification 100.
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Figure 3. Histological and immunohistochemical results for PCL–HA scaffolds loaded with bone marrow cells. (a, d) H&E staining demonstrated that many cell-laden cartilage lacunae were distributed throughout the scaffolds after 10 weeks in culture. (b, e) Safranin O staining showed that GAG was unevenly distributed in the PCL–HA scaffolds. (c, f) Type II collagen staining showed uneven accumulation of type II collagen in the PCL–HA scaffolds. (a–c) Magnification 100; (d–f) magniEcation 400.
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Figure 5. Surgical treatment and macroscopic evaluation of repaired tissues after a 12-h repair period. Osteochondral defects (6 mm 6 mm 3 mm) were created in the trochlear groove of knees. Then, the PCL–HA scaffolds were implanted into the defects (Group 1) (a), microfracture holes were created in the defects (Group 2) (b), or autologous osteochondral plugs were reimplanted into the defects (Group 3) (c). The immediate filling and repair were evaluated in Group 1 (d), Group 2 (e), or Group 3 (f) after the 12-h repair period. Red arrows indicate the interface between the repaired and host tissues.
tissues (both cartilage and bone) was interrupted (Figure 5(f)). At 12 weeks after implantation in vivo, Group 1 demonstrated an imperfectly regenerated cartilage surface (Figure 6(a)); however, the PCL–HA scaffolds had achieved full contact with host tissues and bone ingrowth was observed in the area of implantation (Figure 6(d)). For Group 2, the regenerative cartilage surface was uneven after 12 weeks of repair (Figure 6(b)). In addition, the repair tissues contacted well with host tissues (Figure 6(e)). Group 3 showed that the surface of cartilage was filled with cartilage-like tissues (Figure 6(c)), which differed from the cartilage surface of the control group (Figure 6(g)). Moreover, the
cartilage surfaces of osteochondral plugs were disconnected from that of host cartilage in Group 3 after 12 weeks of in vivo repair (Figure 6(f)).
Histological and immunohistochemical evaluations of repaired tissues At 12 weeks after implantation, both good vertical and lateral integration between PCL–HA scaffolds and host bone were observed in Group 1 (Figure 7(e, m)). However, no obvious cartilage regeneration or GAG accumulation was observed (Figure 7(a, i)). For Group 2, a rough cartilage surface and reduced GAG distribution were detected after 12 weeks of repair
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Figure 6. Macroscopic observation of repaired tissues after 12 weeks of repair. (a, d) Group 1 showed that PCL–HA scaffolds fully contacted host tissues and bone ingrowth was observed, whereas the cartilage surface was incompletely regenerated. (b, e) Group 2 showed that the surface of regenerated cartilage was uneven, but the repaired tissues contacted well with host tissues. (c, f) Group 3 demonstrated that the surface of osteochondral plugs, which differed from those of the control group (g), was disconnected from that of the host cartilage. Red arrows indicate the interface between the repaired and host tissues.
(Figure 7(b, j)). Furthermore, an unstable integration with host tissues was found, suggesting the repaired tissues cannot withstand long-term loading conditions (Figure 7(f, n)). The cartilage layers of Group 3 resembled those of the control group (Figure 7(d, h, l, p)), whereas a decreased distribution of GAG was observed in Group 3 (Figure 7(k, o)). Moreover, the integration between reimplanted cartilage and host cartilage was interrupted at 12 weeks after implantation (Figure 7(c, g, k, o)). The distribution of type II collagen followed a trend similar to that of GAG. There was no obvious accumulation of type II collagen in Group 1 after 12 weeks of repair (Figure 8(a)). However, type I collagen was distinctly detected in the repaired bone layer in Group 1 (Figure 8(i, m)), where its pattern resembled the distribution of type I collagen in the control group (Figure 8(l, p)). Additionally, good integration (vertical and lateral) between PCL–HA scaffolds and host bone was easily detected (Figure 8(e, m)). For Group 2, type II collagen was distributed throughout the repaired tissues (Figure 8(b)), whereas type I collagen was observed in the superficial layer of the repaired tissues (Figure 8(j)). Moreover, a disordered direction for collagen fibers, abnormal bone growth, and unstable integration with host tissues were detected in Group 2 (Figure 8(f, n)). The concentrations of type II and type I collagen in Group 3 (Figure 8(c, k)) were similar to those in the control group (Figure 8(d, h, l, p)). However, the lateral integration between osteochondral plugs and host cartilage was interrupted at 12 weeks after implantation (Figure 8(g, o)).
Discussion In the present study, we fabricated a three-dimensional PCL–HA scaffold using the RP technique (FDM). Then, we investigated the chondrogenic potential of the PCL–HA scaffolds loaded with bone marrow cells in vitro and the influence of PCL–HA scaffolds on osteochondral repair in vivo. Our results showed that PCL–HA scaffolds enhanced chondrogenesis when combined with bone marrow cells. Moreover, the implanted PCL–HA scaffolds improved both vertical and lateral integration with host bone; however, cartilage layers were undesirably regenerated after 12 weeks of repair in vivo. To date, PCL and HA have been widely used in tissue engineering applications.24–27 Shor et al. demonstrated that the composite PCL–HA scaffolds have a higher compressive properties than PCL scaffolds. Moreover, the proliferation and differentiation of fetal bovine osteoblasts on the PCL–HA scaffolds were detected with culture time in vitro.33 Ang and coworkers designed PCL–HA scaffolds with different compositions (100/0, 95/5, 90/10, 80/20, and 70/30 wt%) and investigated the influence of HA content on the mechanical and degradable properties of PCL–HA scaffolds. Their results showed that the degradation rate of PCL–HA scaffolds gradually increases with an increase in HA content. Meanwhile, the compressive strength of PCL–HA scaffolds resembled that of trabecular bone, although the mechanical properties decreased along with the degradation of the scaffolds.34 However, these studies all focused on the application of
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Figure 7. Histological evaluation of repaired tissues. (a–h) H&E staining of the repaired tissues from each group after 12 weeks of in vivo repair. Original magnification 20; detailed magnification 100. (i–p) Safranin O staining of the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. Red arrows indicated excellent both vertical and lateral integration between PCL–HA scaffolds and host bone. White arrows indicate abnormal bone growth and unstable integration with host bone. Black arrows indicate the interrupted lateral integration between osteochondral plugs and host cartilage.
PCL–HA scaffolds for bone tissue engineering. In this study, the porous PCL–HA scaffolds (70/30 wt%) demonstrated good cell viability. Moreover, no obvious degradation of PCL–HA scaffolds, which were loaded with bone marrow cells, was detected after 10 weeks in culture in vitro, suggesting that bone marrow cells proliferated and further differentiated on the PCL–HA scaffolds and eventually prevented the degradation of scaffolds. Currently, only a couple of studies have investigated the chondrogenic potential of PCL–HA scaffolds with controllable structures, fine biocompatibility, and mechanical properties that are critical for cartilage tissue engineering.31,32 Lee et al.31 fabricated a porous composite PCL–HA scaffold that had an average pore diameter of 400 mm and detected cell adhesion and osteochondral regeneration using this scaffold. Further, Lee and associates designed a PCL–HA bioscaffold with interconnecting microchannels for joint regeneration (cartilage layer: 400 mm in diameter; bone layer: 200 mm in diameter). Then, the bioscaffold was infused with TGF-b3–adsorbed collagen hydrogel
and implanted to replace the excised joint. Their findings showed that the entire joint was regenerated without cell implantation.32 In the present study, we fabricated porous PCL–HA scaffolds (average pore diameter of 400 mm) and combined the scaffolds with bone marrow cells, but did not seed precultured cells into the scaffolds. The cultured scaffolds thus resembled the repaired tissues obtained following microfracturebased cartilage repair. Our results showed that many chondrocytes within cartilage lacunae were detected, and cartilage matrix was concentrated in the PCL– HA scaffolds loaded with bone marrow cells after 10 weeks in culture in chondrogenic medium. Moreover, the physical density of cultured PCL–HA scaffolds resembled that of cartilage, but was lower than that of bone. These results indicated that the PCL–HA scaffolds have a potential role in the treatment of microfracture-based cartilage repair. Generally, the repaired tissues obtained following microfracture resembled fibrocartilage and demonstrated inferior mechanical properties compared to normal hyaline cartilage.10 Additionally, lateral
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Figure 8. Immunohistochemical evaluation of repaired tissues. (a–h) Type II collagen staining in the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. (i–p) Type I collagen staining of the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. Red arrows indicate excellent both vertical and lateral integration between PCL–HA scaffolds and host bone. White arrows indicate abnormal bone growth and unstable integration with host bone. Black arrows indicate the interrupted lateral integration between osteochondral plugs and host cartilage.
integration between the implanted cartilage and the surrounding cartilage was limited after AOT.15,16 For this reason, many biomaterials have been developed to improve microfracture-based cartilage repair and integration between implanted cartilage and host cartilage.8,35,36 In this study, we implanted a porous PCL– HA scaffold to repair osteochondral defects. Meanwhile, microfracture and AOT were applied to detect the fibrocartilage repair and the interrupted lateral integration, respectively. Our results showed that the implantation of porous PCL–HA scaffolds enhanced integration (both vertical and lateral) between scaffolds and host bone. Although bone marrow contains some cytokines and growth factors37 and demonstrates paracrine interactions between bone marrow-derived stem cells and other bone marrowderived cells,38 the cartilage surface was not effectively regenerated by 12 weeks after implantation. This may be due to insufficient trophic support from bone marrow components. Thus, the addition of trophic factors as a supplement may be used to achieve excellent cartilage regeneration in future studies.
There exist some limitations in this study. Firstly, we fabricated the PCL–HA scaffolds containing 70 wt% PCL and 30 wt% HA. Future studies will design the PCL–HA scaffolds with different compositions and investigate the effects of the contents of each component (PCL or HA) on cartilage regeneration within PCL–HA scaffolds. Additionally, the PCL–HA scaffolds with a shifted pattern structure can produce compact structures for cell adhesion.28 Thus, PCL–HA scaffolds with different pore sizes and structures can be designed to detect the attachment, proliferation, and differentiation of bone marrow cells in the scaffolds. Finally, PCL–HA scaffolds loaded with bone marrow cells were prepared and implanted in osteochondral defects. The effect of precultured cells seeded within PCL–HA scaffolds on osteochondral repair will be further evaluated in future studies. Taken together, our results show that three-dimensional porous PCL–HA scaffolds loaded with bone marrow cells can enhance chondrogenesis in vitro and the implantation of PCL–HA scaffolds for osteochondral repair can facilitate both vertical and lateral
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10 integration between the scaffold and host bone. Nevertheless, the surface of cartilage was not effectively regenerated in the present study. Scaffold loading with trophic factors or precultured cells for accelerated osteochondral repair requires further investigation.
Authors’ note Bo Wei and Qingqiang Yao contributed equally to this work. Acknowledgment We thank Mrs. Yuling Peng for her logistical support.
Declaration of conflicting interests None declared.
Funding This work was supported by the National Natural Science Foundation of China (No. 81171745) and the Orthopedic Clinical Medical Center of Nanjing City, China.
References 1. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg 2011; 77: 709–726. 2. Ahmed TA and Hincke MT. Strategies for articular cartilage lesion repair and functional restoration. Tissue Eng Part B Rev 2010; 16: 305–329. 3. Steadman JR, Rodkey WG and Rodrigo JJ. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res 2001; 391: S362–S369. 4. Karataglis D, Green MA and Learmonth DJ. Autologous osteochondral transplantation for the treatment of chondral defects of the knee. Knee 2006; 13: 32–35. 5. Minas T. Autologous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop Relat Res 2001; 391: S349–S361. 6. Magnuson PB. The classic: joint debridement: surgical treatment of degenerative arthritis. Clin Orthop Relat Res 1974; 101: 4–12. 7. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cellbased repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 1994; 76: 579–592. 8. Gomoll AH. Microfracture and augments. J Knee Surg 2012; 25: 9–15. 9. Mithoefer K, Williams RJ 3rd, Warren RF, et al. The microfracture technique for the treatment of articular cartilage lesions in the knee. A prospective cohort study. J Bone Joint Surg Am 2005; 87: 1911–1920. 10. Kaul G, Cucchiarini M, Remberger K, et al. Failed cartilage repair for early osteoarthritis defects: a biochemical, histological and immunohistochemical analysis of the repair tissue after treatment with marrow-stimulation techniques. Knee Surg Sports Traumatol Arthrosc 2012; 20: 2315–2324.
Journal of Biomaterials Applications 0(0) 11. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cartil 2002; 10: 432–463. 12. Shapiro F, Koide S and Glimcher MJ. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg Am 1993; 75: 532–553. 13. Kreuz PC, Erggelet C, Steinwachs MR, et al. Is microfracture of chondral defects in the knee associated with different results in patients aged 40 years or younger. Arthroscopy 2006; 22: 1180–1186. 14. Hangody L, Kish G, Ka´rpa´ti Z, et al. Mosaicplasty for the treatment of articular cartilage defects: application in clinical practice. Orthopedics 1998; 21: 751–756. 15. Horas U, Pelinkovic D, Herr G, et al. Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial. J Bone Joint Surg Am 2003; 85-A: 185–192. 16. Khan IM, Gilbert SJ, Singhrao SK, et al. Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review. Eur Cell Mater 2008; 16: 26–39. 17. Zhang Q, Lu H, Kawazoe N, et al. Pore size effect of collagen scaffolds on cartilage regeneration. Acta Biomater 2014; 10: 2005–2013. 18. Shanmugasundaram S, Chaudhry H and Arinzeh TL. Microscale versus nanoscale scaffold architecture for mesenchymal stem cell chondrogenesis. Tissue Eng Part A 2011; 17: 831–840. 19. Nuernberger S, Cyran N, Albrecht C, et al. The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts. Biomaterials 2011; 32: 1032–1040. 20. Xu Y, Xu GY, Tang C, et al. Preparation and characterization of bone marrow mesenchymal stem cell-derived extracellular matrix scaffolds. J Biomed Mater Res B Appl Biomater 2014 Jul 5. 21. Wei B, Jin C, Xu Y, et al. Chondrogenic differentiation of marrow clots after microfracture with BMSC-derived ECM scaffold in vitro. Tissue Eng Part A 2014; 20: 2646–2655. 22. Yang S, Leong KF, Du Z, et al. The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng 2002; 8: 1–11. 23. Yeong WY, Chua CK, Leong KF, et al. Rapid prototyping in tissue engineering: challenges and potential. Trends Biotechnol 2004; 22: 643–652. 24. Hutmacher DW, Schantz T, Zein I, et al. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mater Res 2001; 55: 203–216. 25. Cao T, Ho KH and Teoh SH. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling. Tissue Eng 2003; 9: S103–S112. 26. Oonishi H. Orthopaedic applications of hydroxyapatite. Biomaterials 1991; 12: 171–178. 27. Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005; 4: 518–524.
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Original article
Three-dimensional polycaprolactone– hydroxyapatite scaffolds combined with bone marrow cells for cartilage tissue engineering
Journal of Biomaterials Applications 0(0) 1–11 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215575762 jba.sagepub.com
Bo Wei1,2,3, Qingqiang Yao1,2,3, Yang Guo1,2,3, Fengyong Mao1,2, Shuai Liu1,2, Yan Xu1,2,3 and Liming Wang1,2,3
Abstract The goal of this study was to investigate the chondrogenic potential of three-dimensional polycaprolactone–hydroxyapatite (PCL–HA) scaffolds loaded with bone marrow cells in vitro and the effect of PCL–HA scaffolds on osteochondral repair in vivo. Here, bone marrow was added to the prepared PCL–HA scaffolds and cultured in chondrogenic medium for 10 weeks. Osteochondral defects were created in the trochlear groove of 29 knees in 17 New Zealand white rabbits, which were then divided into four groups that underwent: implantation of PCL–HA scaffolds (left knee, n ¼ 17; Group 1), microfracture (right knee, n ¼ 6; Group 2), autologous osteochondral transplantation (right knee, n ¼ 6; Group 3), and no treatment (right knee, n ¼ 5; Control). Extracellular matrix produced by bone marrow cells covered the surface and filled the pores of PCL–HA scaffolds after 10 weeks in culture. Moreover, many cell-laden cartilage lacunae were observed, and cartilage matrix was concentrated in the PCL–HA scaffolds. After a 12-week repair period, Group 1 showed excellent vertical and lateral integration with host bone, but incomplete cartilage regeneration and matrix accumulation. An uneven surface of regenerated cartilage and reduced distribution of cartilage matrix were observed in Group 2. In addition, abnormal bone growth and unstable integration between repaired and host tissues were detected. For Group 3, the integration between transplanted and host cartilage was interrupted. Our findings indicate that the PCL–HA scaffolds loaded with bone marrow cells improved chondrogenesis in vitro and implantation of PCL–HA scaffolds for osteochondral repairenhanced integration with host bone. However, cartilage regeneration remained unsatisfactory. The addition of trophic factors or the use of precultured cell–PCL–HA constructs for accelerated osteochondral repair requires further investigation. Keywords Polycaprolactone, hydroxyapatite, bone marrow cells, cartilage tissue engineering, three dimensional
Introduction Healthy articular cartilage has low vascularity, which endows it with limited ability to respond to injury. The primary aim of cartilage repair is to restore the structure and function of damaged cartilage.1,2 Many surgical interventions are widely available for treating damaged cartilage.3–7 Among them, microfracture is highly appreciated because of its minimal invasiveness, simplicity, and low cost.3,8,9 However, the repaired tissues are composed mainly of fibrocartilage, which has inferior biomechanical properties compared to hyaline cartilage.10 Eventually, discontinuous lateral integration, abnormal bone growth, and degenerative cartilage
1 Department of Orthopaedic Surgery, Nanjing First Hospital, Nanjing Medical University, Nanjing, China 2 Cartilage Regeneration Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China 3 China-Korea United Cell Therapy Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
Corresponding author: Liming Wang, Department of Orthopedic Surgery, Nanjing First Hospital, Nanjing Medical University, PO Box 210006, Nanjing 210006, Jiangsu, People’s Republic of China. Email: [email protected]
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develop.11,12 Moreover, the clinical outcomes after microfracture-based cartilage repair of the knee are age dependent, and patients aged 40 years or younger with lesions on the femoral condyles experience the best outcomes.13 Additionally, autologous osteochondral transplantation (AOT) has been widely used because it can achieve fine vertical fixation, followed by bone healing and structural reconstitution.4,14 However, the lateral integration between the osteochondral plugs and the surrounding host cartilage is limited, which can lead to poor mechanical properties at the interface regions and eventually cause cartilage degeneration.15,16 Therefore, various biomaterial scaffolds have been investigated to improve cartilage repair. Generally, an ideal scaffold for cartilage repair should demonstrate mechanical properties that resemble those of native cartilage, promote the concentration of cartilage-like extracellular matrix (ECM), and integrate well with the surrounding host tissues.2 Moreover, scaffolds fabricated for cartilage tissue engineering usually possess a porous structure that facilitates cell adhesion, proliferation, and differentiation.17–19 Previously, we developed a porous bone marrow mesenchymal stem cell– derived (BMSC–d) ECM scaffold and investigated the chondrogenesis within the BMSC–dECM scaffold loaded with bone marrow cells.20,21 However, the mechanical properties of the BMSC–dECM scaffold are inferior to those of native cartilage, suggesting that the scaffolds cannot withstand long-term load bearing during the repair of articular cartilage. Recently, rapid prototyping (RP) techniques have been developed for fabricating scaffolds with controllable microstructures and properties.22,23 Thus, scaffolds with controlled porosity and mechanical strength can be obtained using RP techniques. Polycaprolactone (PCL) is a nontoxic, slow degrading, and tissue compatible polymer that can be used as a biomaterial for tissue engineering applications.24,25 Hydroxyapatite (HA), which demonstrates chemical (a)
(b)
constituents resembling the mineral components of bone, is widely considered suitable for bone tissue engineering.26,27 Furthermore, many groups have investigated blends of PCL and HA as scaffolds offering improved mechanical properties, cell adhesion and proliferation, and osteoconductivity.28–30 However, rare studies have examined the chondrogenic potential of a porous PCL–HA scaffold with controllable pore size, fine biocompatibility, and mechanical properties critical for cartilage tissue engineering.31,32 Thus, in the present study, we tested our hypothesis that a three-dimensional PCL–HA scaffold loaded with bone marrow cells can enhance cartilage regeneration in vitro and improve osteochondral repair in vivo.
Materials and methods Fabrication of three-dimensional PCL–HA scaffolds with controllable microstructure The three-dimensional properties of PCL–HA scaffolds were designed using computer-aided design software. First, 70 wt% PCL (molecular weight65,000, Sigma Chemical Co., St. Louis, MO, USA) was mixed with 30 wt% HA power (Sigma Chemical Co.) in a slurry at 120%. The PCL–HA scaffolds were fabricated using a three-dimensional layer-by-layer fused deposition modeling (FDM) 3000 system (Stratasys Inc., USA). The obtained PCL–HA scaffolds had a diameter of 6 mm, a thickness of 3 mm, and an average pore size of 400 mm (Figure 1(a, b)). The fabricated PCL–HA scaffolds were sterilized using ethylene oxide for subsequent in vitro chondrogenesis and in vivo implantation experiments.
In vitro chondrogenesis in PCL–HA scaffolds loaded with bone marrow cells All animal experiments were approved by the Institutional Review Board and the Animal Research (c)
Figure 1. Gross and microscopic observation of and cell viability within polycaprolactone/hydroxyapatite (PCL–HA) scaffolds. (a) The porous PCL–HA scaffolds consisting of 70 wt% PCL and 30 wt% HA were fabricated using a rapid prototyping technique. (b) Scanning electron microscopy (SEM) images showed that the porous PCL–HA scaffolds had an average pore size of 400 mm. MagniEcation25. (c) Confocal microscopy images demonstrated most live cells emitted green fluorescence, and few dead cells were labeled with red fluorescence after seven days in culture. Magnification100.
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Wei et al. Committee of Nanjing Medical University. Bone marrow samples were aseptically obtained from the posterior superior iliac spine of 17 female New Zealand white rabbits (aged four to six months, weighting 3.5–4.0 kg) via the bone marrow puncture procedure. Then, the prepared PCL–HA scaffolds were immersed in the collected bone marrow for 40 min at room temperature to ensure good contact between PCL–HA scaffolds and bone marrow cells. The obtained composite scaffolds were immersed in chondrogenic medium (high-glucose Dulbecco’s modified Eagle’s medium [Gibco, Australia], 10% fetal bovine serum [Gibco, Australia], 1% penicillin–streptomycin, 40 mg/ml L-proline, 100 mg/ml sodium pyruvate, 37.5 mg/ml L-ascorbic acid 2-phosphate, 6.25 mg/ml bovine insulin, 6.25 mg/ml transferrin, 6.25 mg/ml selenous acid, 5.33 mg/ml linoleic acid, 100 nM dexamethasone, and 10 ng/ml transforming growth factor-b3 [TGF-b3; PeproTech, Rocky Hill, NJ, USA])21 for 10 weeks in vitro.
Cell viability, gross observation, density, and microstructure Cell viability in the PCL–HA scaffolds was evaluated after seven days in culture using the Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit (Molecular Probes/Invitrogen, Eugene, OR, USA). Briefly, samples were collected and washed with phosphate-buffered saline, incubated in the combined LIVE/ DEAD assay reagents for 30 min at room temperature, and then observed using a confocal microscope (Leica TCS SP2 Confocal Microscope; Leica, Mannheim, Germany). Live cells are marked with green fluorescence, and dead cells are labeled with red fluorescence. The macroscopic images of PCL–HA scaffolds were observed after 10 weeks in vitro. Then, the PCL–HA scaffolds were scanned to detect the calcification of matrix via micro-computed tomography (micro-CT; Hiscan MCT-1108, Nanjing, China) with a scanning width of 50 mm. The surface of PCL–HA scaffolds was positioned vertical to the axis of the ray beam. Meanwhile, a native osteochondral plug was scanned as a control. Additionally, the microstructures of both surface and transverse sections of the PCL–HA scaffolds were evaluated by scanning electron microscopy (SEM; Hitachi S4800, Tokyo, Japan).
Histology and immunohistochemistry After 10 weeks in culture, the harvested PCL–HA scaffolds were fixed in 10% neutral formalin for 24 h, decalcified in 5% nitric acid for 48 h, dehydrated using a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections (5 mm in thickness) were stained
3 with hematoxylin–eosin (H&E) and Safranin-O/FastGreen (Safranin O), separately. For immunohistochemical staining, sections were sequentially exposed to proteinase K for 30 min, 3% hydrogen peroxide in methanol for 10 min, 10% goat serum for 30 min, and mouse antitype II collagen monoclonal antibody (Novus Biologicals, Littleton, CO, USA) for 90 min, followed by goat anti-mouse secondary antibody (Nanjing KeyGen Biotech. Co., Ltd., Jiangsu, China) for 30 min and 3,30 -diaminobenzidine tetrahydrochloride solution for 3–5 min. Then, the sections were counterstained with Mayer’s hematoxylin (Sigma) for 8 min and mounted with neutral balsam.
Biochemical analysis After 1 and 10 weeks in culture, the total DNA content of PCL–HA scaffolds was measured using a Quit-iT dsDNA kit (Molecular Probes/Invitrogen, Eugene, OR, USA) (n ¼ 3). Briefly, the samples were digested with papain and reacted with Hoechst dye 33258 for 30 min. Fluorescence intensity was detected with a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) with excitation at 360 nm and emission at 460 nm. DNA from salmon testes (Sigma) was used to create a standard curve. The glycosaminoglycan (GAG) content of PCL–HA scaffolds was measured using a BlyscanTM sulfated GAG assay kit (Biocolor, Newtonabbey, UK) (n ¼ 3). In brief, a papain-digested solution was mixed with Blyscan dye reagent and centrifuged at 12,000 r/min for 10 min. The nonbound GAG dye was then removed, and dissociation reagent was added. A microplate reader (Bio-Rad Laboratories) was used to measure absorbance at 656 nm. The collagen content of PCL–HA scaffolds was quantified using a Sircol Collagen Assay Kit (Biocolor) (n ¼ 3). Briefly, the samples were digested with pepsin, mixed with Sircol dye reagent, and centrifuged at 12,000 r/min for 10 min. The deposit was then washed with acid–salt wash reagent and dissolved in alkali reagent. A microplate reader (Bio-Rad Laboratories) was used to measure the absorbance at 555 nm.
Surgical treatment and grouping A total of 17 New Zealand white rabbits, which were used for bone marrow collection to assay in vitro chondrogenesis, were anesthetized by intramuscular administration of ketamine hydrochloride (15 mg/kg). Osteochondral defects (6 mm 6 mm 3 mm) were created in the trochlear groove of 29 knees. PCL–HA scaffolds unseeded with cells were implanted into the defects of left knees (n ¼ 17; Group 1). Microfracture holes were created in the defects of right knees (n ¼ 6;
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4 Group 2). Osteochondral plugs, which were harvested from the osteochondral defects, were reimplanted into the defects of right knees (n ¼ 6; Group 3). Meanwhile, untreated knees were designated as controls (n ¼ 5; Control). Twelve hours after implantation, two rabbits (one with the left knee in Group 1 and the right knee in Group 2, and another with the left knee in Group 1 and the right knee in Group 3) were sacrificed using an overdose of sodium pentobarbital to observe the immediate filling. At 12 weeks after implantation, the remaining rabbits were sacrificed for further analysis.
Macroscopic evaluation, histological analysis, and immunohistochemical staining of repaired tissues Knee joints (distal femurs) were harvested, and the gross morphology (including that of both surface and longitudinal sections) of repaired tissues was assessed after 12 weeks of repair in vivo. Then, the samples were fixed in 10% neutral formalin for 24 h, decalcified in 5% nitric acid for 96 h, and embedded in paraffin. Sections (5 mm in thickness) were stained with H&E and Safranin O to evaluate cell morphology, proteoglycan distribution, and integration with surrounding host tissues (including both vertical and lateral integration). Separate sections (5 mm in thickness) were incubated with mouse antitype II collagen monoclonal antibody (Novus Biologicals, Littleton, CO, USA) and mouse antitype I collagen antibody (Sigma–Aldrich, St. Louis, MO, USA) for 90 min at 37 C to observe type II and type I collagen production, respectively.
Statistical analysis All data are expressed as the mean standard deviation. The unpaired Student’s t-test was used to compare the differences in the total DNA content, GAG content, and collagen content of PCL–HA scaffolds between 1 and 10 weeks in culture. The level of statistical significance was set at P < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software (version 13.0; SPSS Inc., Chicago, IL, USA).
Results Cell viability and macroscopic and microscopic observations of PCL–HA scaffolds loaded with bone marrow cells After seven days in culture, bone marrow cell viability in the PCL–HA scaffolds was evaluated using confocal microscopy. Many live cells emitted green fluorescence and were distributed on the fibers and around the pores of PCL–HA scaffolds. Meanwhile, few dead cells
Journal of Biomaterials Applications 0(0) emitting red fluorescence were unevenly distributed in the PCL–HA scaffolds (Figure 1(c)). After 10 weeks in culture, many hyaline-like tissues had formed on the surface of PCL–HA scaffolds, particularly in the peripheral regions of the scaffolds. Moreover, the pores of scaffolds were uniformly filled (Figure 2(a)). Micro-CT images showed that the physical density of PCL–HA scaffolds resembled that of cartilage, but were lower than that of bone (Figure 2(b, c)). Furthermore, SEM images demonstrated that both the surface and transverse sections of PCL–HA scaffolds were evenly covered with ECM, indicating that proliferation and differentiation of marrow cells occurred in the PCL– HA scaffolds (Figure 2(d, e)).
Histological, immunohistochemical, and biochemical analyses of PCL–HA scaffolds loaded with bone marrow cells After 10 weeks in culture, H&E staining of PCL–HA scaffolds showed that many cell-laden cartilage lacunae were distributed throughout the scaffolds (Figure 3(a,d)). Safranin O and type II collagen staining demonstrated that GAG and type II collagen were unevenly concentrated in the fibers of PCL–HA scaffolds (Figure 3(b, c, e, f)). Furthermore, the contents of DNA, GAG, and collagen in PCL–HA scaffolds were detected after 1 and 10 weeks in culture, individually. The average total DNA content at 10 weeks (2.26 0.17 mg/mg) was significantly higher than that at one week (0.96 0.08 mg/mg; P < 0.001; Figure 4(a)). Similarly, the contents of GAG and collagen at 10 weeks (27.19 0.90 mg/mg GAG; 50.77 2.83 mg/mg collagen) were significantly higher than those at one week (5.35 0.23 mg/mg GAG; 8.38 0.68 mg/mg collagen; P < 0.001; Figure 4(b, c)).
Macroscopic evaluation of repaired tissues No obvious effusions or inflammative reactions were detected in the knee joints of all rabbits. After the PCL–HA scaffolds were implanted into the osteochondral defects (Figure 5(a)), Group 1 showed that the scaffolds were firmly fixed in the defects and well contacted by marrow clots by 12 h following microfracture (Figure 5(d)). After microfractures were created in the knee joints (Figure 5(b)), Group 2 demonstrated that osteochondral defects filled with marrow clots after 12 h. Moreover, the repair tissues were integrated well with surrounding tissues (Figure 5(e)). After autologous osteochondral plugs were retransplanted into the osteochondral defects (Figure 5(c)), Group 3 showed that the cartilage surface of plugs was aligned with the surface of the host cartilage after 12 h. However, the integration between osteochondral plugs and host
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Figure 2. Macroscopic and microscopic evaluations of PCL–HA scaffolds loaded with bone marrow cells after 10 weeks in culture. (a) Gross observation demonstrated that hyaline-like tissues had formed over the surface of PCL–HA scaffolds. (b, c) Micro-CT images revealed that the physical density of PCL–HA scaffolds resembled that of cartilage, but was lower than that of bone. (d, e) SEM images showed that both the surface and transverse sections of PCL–HA scaffolds were evenly distributed with extracellular matrix (ECM). Magnification 100.
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Figure 3. Histological and immunohistochemical results for PCL–HA scaffolds loaded with bone marrow cells. (a, d) H&E staining demonstrated that many cell-laden cartilage lacunae were distributed throughout the scaffolds after 10 weeks in culture. (b, e) Safranin O staining showed that GAG was unevenly distributed in the PCL–HA scaffolds. (c, f) Type II collagen staining showed uneven accumulation of type II collagen in the PCL–HA scaffolds. (a–c) Magnification 100; (d–f) magniEcation 400.
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Figure 5. Surgical treatment and macroscopic evaluation of repaired tissues after a 12-h repair period. Osteochondral defects (6 mm 6 mm 3 mm) were created in the trochlear groove of knees. Then, the PCL–HA scaffolds were implanted into the defects (Group 1) (a), microfracture holes were created in the defects (Group 2) (b), or autologous osteochondral plugs were reimplanted into the defects (Group 3) (c). The immediate filling and repair were evaluated in Group 1 (d), Group 2 (e), or Group 3 (f) after the 12-h repair period. Red arrows indicate the interface between the repaired and host tissues.
tissues (both cartilage and bone) was interrupted (Figure 5(f)). At 12 weeks after implantation in vivo, Group 1 demonstrated an imperfectly regenerated cartilage surface (Figure 6(a)); however, the PCL–HA scaffolds had achieved full contact with host tissues and bone ingrowth was observed in the area of implantation (Figure 6(d)). For Group 2, the regenerative cartilage surface was uneven after 12 weeks of repair (Figure 6(b)). In addition, the repair tissues contacted well with host tissues (Figure 6(e)). Group 3 showed that the surface of cartilage was filled with cartilage-like tissues (Figure 6(c)), which differed from the cartilage surface of the control group (Figure 6(g)). Moreover, the
cartilage surfaces of osteochondral plugs were disconnected from that of host cartilage in Group 3 after 12 weeks of in vivo repair (Figure 6(f)).
Histological and immunohistochemical evaluations of repaired tissues At 12 weeks after implantation, both good vertical and lateral integration between PCL–HA scaffolds and host bone were observed in Group 1 (Figure 7(e, m)). However, no obvious cartilage regeneration or GAG accumulation was observed (Figure 7(a, i)). For Group 2, a rough cartilage surface and reduced GAG distribution were detected after 12 weeks of repair
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Figure 6. Macroscopic observation of repaired tissues after 12 weeks of repair. (a, d) Group 1 showed that PCL–HA scaffolds fully contacted host tissues and bone ingrowth was observed, whereas the cartilage surface was incompletely regenerated. (b, e) Group 2 showed that the surface of regenerated cartilage was uneven, but the repaired tissues contacted well with host tissues. (c, f) Group 3 demonstrated that the surface of osteochondral plugs, which differed from those of the control group (g), was disconnected from that of the host cartilage. Red arrows indicate the interface between the repaired and host tissues.
(Figure 7(b, j)). Furthermore, an unstable integration with host tissues was found, suggesting the repaired tissues cannot withstand long-term loading conditions (Figure 7(f, n)). The cartilage layers of Group 3 resembled those of the control group (Figure 7(d, h, l, p)), whereas a decreased distribution of GAG was observed in Group 3 (Figure 7(k, o)). Moreover, the integration between reimplanted cartilage and host cartilage was interrupted at 12 weeks after implantation (Figure 7(c, g, k, o)). The distribution of type II collagen followed a trend similar to that of GAG. There was no obvious accumulation of type II collagen in Group 1 after 12 weeks of repair (Figure 8(a)). However, type I collagen was distinctly detected in the repaired bone layer in Group 1 (Figure 8(i, m)), where its pattern resembled the distribution of type I collagen in the control group (Figure 8(l, p)). Additionally, good integration (vertical and lateral) between PCL–HA scaffolds and host bone was easily detected (Figure 8(e, m)). For Group 2, type II collagen was distributed throughout the repaired tissues (Figure 8(b)), whereas type I collagen was observed in the superficial layer of the repaired tissues (Figure 8(j)). Moreover, a disordered direction for collagen fibers, abnormal bone growth, and unstable integration with host tissues were detected in Group 2 (Figure 8(f, n)). The concentrations of type II and type I collagen in Group 3 (Figure 8(c, k)) were similar to those in the control group (Figure 8(d, h, l, p)). However, the lateral integration between osteochondral plugs and host cartilage was interrupted at 12 weeks after implantation (Figure 8(g, o)).
Discussion In the present study, we fabricated a three-dimensional PCL–HA scaffold using the RP technique (FDM). Then, we investigated the chondrogenic potential of the PCL–HA scaffolds loaded with bone marrow cells in vitro and the influence of PCL–HA scaffolds on osteochondral repair in vivo. Our results showed that PCL–HA scaffolds enhanced chondrogenesis when combined with bone marrow cells. Moreover, the implanted PCL–HA scaffolds improved both vertical and lateral integration with host bone; however, cartilage layers were undesirably regenerated after 12 weeks of repair in vivo. To date, PCL and HA have been widely used in tissue engineering applications.24–27 Shor et al. demonstrated that the composite PCL–HA scaffolds have a higher compressive properties than PCL scaffolds. Moreover, the proliferation and differentiation of fetal bovine osteoblasts on the PCL–HA scaffolds were detected with culture time in vitro.33 Ang and coworkers designed PCL–HA scaffolds with different compositions (100/0, 95/5, 90/10, 80/20, and 70/30 wt%) and investigated the influence of HA content on the mechanical and degradable properties of PCL–HA scaffolds. Their results showed that the degradation rate of PCL–HA scaffolds gradually increases with an increase in HA content. Meanwhile, the compressive strength of PCL–HA scaffolds resembled that of trabecular bone, although the mechanical properties decreased along with the degradation of the scaffolds.34 However, these studies all focused on the application of
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Figure 7. Histological evaluation of repaired tissues. (a–h) H&E staining of the repaired tissues from each group after 12 weeks of in vivo repair. Original magnification 20; detailed magnification 100. (i–p) Safranin O staining of the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. Red arrows indicated excellent both vertical and lateral integration between PCL–HA scaffolds and host bone. White arrows indicate abnormal bone growth and unstable integration with host bone. Black arrows indicate the interrupted lateral integration between osteochondral plugs and host cartilage.
PCL–HA scaffolds for bone tissue engineering. In this study, the porous PCL–HA scaffolds (70/30 wt%) demonstrated good cell viability. Moreover, no obvious degradation of PCL–HA scaffolds, which were loaded with bone marrow cells, was detected after 10 weeks in culture in vitro, suggesting that bone marrow cells proliferated and further differentiated on the PCL–HA scaffolds and eventually prevented the degradation of scaffolds. Currently, only a couple of studies have investigated the chondrogenic potential of PCL–HA scaffolds with controllable structures, fine biocompatibility, and mechanical properties that are critical for cartilage tissue engineering.31,32 Lee et al.31 fabricated a porous composite PCL–HA scaffold that had an average pore diameter of 400 mm and detected cell adhesion and osteochondral regeneration using this scaffold. Further, Lee and associates designed a PCL–HA bioscaffold with interconnecting microchannels for joint regeneration (cartilage layer: 400 mm in diameter; bone layer: 200 mm in diameter). Then, the bioscaffold was infused with TGF-b3–adsorbed collagen hydrogel
and implanted to replace the excised joint. Their findings showed that the entire joint was regenerated without cell implantation.32 In the present study, we fabricated porous PCL–HA scaffolds (average pore diameter of 400 mm) and combined the scaffolds with bone marrow cells, but did not seed precultured cells into the scaffolds. The cultured scaffolds thus resembled the repaired tissues obtained following microfracturebased cartilage repair. Our results showed that many chondrocytes within cartilage lacunae were detected, and cartilage matrix was concentrated in the PCL– HA scaffolds loaded with bone marrow cells after 10 weeks in culture in chondrogenic medium. Moreover, the physical density of cultured PCL–HA scaffolds resembled that of cartilage, but was lower than that of bone. These results indicated that the PCL–HA scaffolds have a potential role in the treatment of microfracture-based cartilage repair. Generally, the repaired tissues obtained following microfracture resembled fibrocartilage and demonstrated inferior mechanical properties compared to normal hyaline cartilage.10 Additionally, lateral
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Figure 8. Immunohistochemical evaluation of repaired tissues. (a–h) Type II collagen staining in the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. (i–p) Type I collagen staining of the repaired tissues from each group after 12 weeks of repair. Original magnification 20; detailed magnification 100. Red arrows indicate excellent both vertical and lateral integration between PCL–HA scaffolds and host bone. White arrows indicate abnormal bone growth and unstable integration with host bone. Black arrows indicate the interrupted lateral integration between osteochondral plugs and host cartilage.
integration between the implanted cartilage and the surrounding cartilage was limited after AOT.15,16 For this reason, many biomaterials have been developed to improve microfracture-based cartilage repair and integration between implanted cartilage and host cartilage.8,35,36 In this study, we implanted a porous PCL– HA scaffold to repair osteochondral defects. Meanwhile, microfracture and AOT were applied to detect the fibrocartilage repair and the interrupted lateral integration, respectively. Our results showed that the implantation of porous PCL–HA scaffolds enhanced integration (both vertical and lateral) between scaffolds and host bone. Although bone marrow contains some cytokines and growth factors37 and demonstrates paracrine interactions between bone marrow-derived stem cells and other bone marrowderived cells,38 the cartilage surface was not effectively regenerated by 12 weeks after implantation. This may be due to insufficient trophic support from bone marrow components. Thus, the addition of trophic factors as a supplement may be used to achieve excellent cartilage regeneration in future studies.
There exist some limitations in this study. Firstly, we fabricated the PCL–HA scaffolds containing 70 wt% PCL and 30 wt% HA. Future studies will design the PCL–HA scaffolds with different compositions and investigate the effects of the contents of each component (PCL or HA) on cartilage regeneration within PCL–HA scaffolds. Additionally, the PCL–HA scaffolds with a shifted pattern structure can produce compact structures for cell adhesion.28 Thus, PCL–HA scaffolds with different pore sizes and structures can be designed to detect the attachment, proliferation, and differentiation of bone marrow cells in the scaffolds. Finally, PCL–HA scaffolds loaded with bone marrow cells were prepared and implanted in osteochondral defects. The effect of precultured cells seeded within PCL–HA scaffolds on osteochondral repair will be further evaluated in future studies. Taken together, our results show that three-dimensional porous PCL–HA scaffolds loaded with bone marrow cells can enhance chondrogenesis in vitro and the implantation of PCL–HA scaffolds for osteochondral repair can facilitate both vertical and lateral
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Authors’ note Bo Wei and Qingqiang Yao contributed equally to this work. Acknowledgment We thank Mrs. Yuling Peng for her logistical support.
Declaration of conflicting interests None declared.
Funding This work was supported by the National Natural Science Foundation of China (No. 81171745) and the Orthopedic Clinical Medical Center of Nanjing City, China.
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