Drs Shi, Zhang, Zhu, Pi, and Ao are from the Institute of Sports Medicine, Dr Zeng is from the Department of Diagnostic Radiology, Peking University Third Hospital, and Dr Zhou is from the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
Drs Shi, Zhang, Zeng, Zhu, Pi, Zhou, and Ao have no relevant financial relationships to disclose.
This study was supported by the National Natural Science Foundation of China (81071474); the Program for Changjiang Scholars and Innovative Research Team in University (BMU2009129-112); and the Research Fund for the Doctoral Program of Higher Education of China (20100001110086).
Correspondence should be addressed to: Yingfang Ao, MD (firstname.lastname@example.org), and Chunyan Zhou, PhD (email@example.com).
Focal full-thickness articular cartilage defects are among the most common injuries in orthopedics and sports medicine. They may result in pain, swelling, hopping, and eventually osteoarthritis.1 The worldwide prevalence of cartilage injury was 60% among patients having undergone knee arthroscopy.2 Due to the limited regeneration capacity of chondrocytes, articular cartilage repair remains a challenging.
Microfracture is one of the first-line treatments for cartilage defects smaller than 2 cm2. This method is technically less demanding and more cost-effective, with lower complication rates and less postoperative pain compared with more invasive procedures.3 The procedure creates multiple holes that penetrate the subchondral bone, allowing marrow and blood to aggregate into the defect sites, along with bone marrow mesenchymal stem cells (marrow stem cells) that differentiate into chondrocytes. However, microfracture only regenerates fibrocartilage instead of hyaline cartilage. In addition, although microfracture provides good short- and mid-term results,4 some studies have reported that the regenerated tissue deteriorated over the long term, and patients could not return to their previous sports levels.5,6
Cartilage tissue engineering provides a new option for articular cartilage defect repair. Several studies have applied culture-expanded marrow stem cells or autologous chondrocytes for cartilage repair with or with no scaffolds in animal experiments or clinical applications.7–11 Matrix-induced chondrocyte implantation is a tissue engineering method used in clinical applications. Although these treatments produce good short-term results, weaknesses exist: the procedures are complicated to perform; patients must undergo 2 surgeries—autologous cells collection and reimplantation after cell expansion; and in vitro cell culture process carries a risk of microbiological contamination. Another concern is donor site morbidity, usually including nonweight-bearing and nonarticulating areas, such as the superomedial edge of the femoral condyle, superolateral edge of the femoral condyle, or the lateral intercondylar notch.8 Therefore, the purpose of this study was to find a 1-step repair method that reduced pain, decreased infection risks, and could be performed more confidently.
In a previous study, the authors proved that combining microfracture with a drilled decalcified cortical bone matrix produced a more marked effect on cartilage repair than combining microfracture with implanting undrilled decalcified cortical bone matrix.12 The authors hypothesized that the drilling holes provided a 3-dimensional residence for stem cells, and that stem cell niches played a significant role in the repair.
Recently, stem cell niches have received much attention. The bone marrow stem cell niche is the microenvironment in the bone marrow for stem cell self-renewal, proliferation, differentiation, mobilization, and homing, which was composed of stem cells, adhesion molecules, extracellular matrix components, growth factors, cytokines, and physiochemical nature.1.13,14 Considering the stem cell niche, the authors used microfracture to produce a microenvironment and provide endogenous stem cells instead of exogenous cells.
In the current study, a 1-step cartilage repair procedure was performed to repair articular cartilage defects in a rabbit model, integrating the in situ autologous bone marrow stem cells produced by microfracture (instead of exogenous stem cells) with cell-free poly(L-lactic-co-glycolic acid) (PLLGA) scaffolds, which provided an appropriate physical support and residence for marrow stem cell growth. The authors evaluated whether this novel method could be an effective option for cartilage repair. This study comprised in vitro and in vivo parts. Studies in vitro detected whether cell-free PLLGA scaffolds could support marrow stem cell adhesion, growth, and proliferation. In the in vivo study, microfracture and cell-free PLLGA scaffold (not the marrow stem cells-PLLGA composition) implantation were applied in 1 surgery to evaluate the effects of cartilage repair.
Materials and Methods
PLLGA scaffolds (Synthecon, Houston, Texas) were composed of polylactic and polyglycolic acid in a ratio of 90/10 with a thickness of 0.8 mm and a density of 79 mg/cm3. The microstructure of the scaffolds was observed by scanning electronic microscopy (SEM).
The male Japanese white rabbits (age, 3–4 months; weight, 3–3.5 kg) used in this study were obtained from Beijing Animal Administration Center. All animal experiments were approved by the Animal Care and Use Committee of Peking University, following the Guide for the Care and Use of Laboratory Animals.15
Cell Proliferation and Morphology on Scaffolds
Marrow stem cells were isolated from the femoral and tibial marrow of rabbits under sterile conditions and were cultured and subcultured in 90% Dulbecco’s Modified Eagle Media with low glucose (GIBCO, Gaithersburg, Maryland) and supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) at 37°C in a humidified incubator containing 5% CO2. Marrow stem cells of passage 1 were seeded on the sterilized PLLGA scaffolds (diameter, 4.5 mm; thickness, 1 mm) at a density of 1.0×106 cells/mL. The marrow stem cells–scaffold complex was cultured on a 96-well plate with a 200 μL culture medium. The cells on the marrow stem cells–PLLGA composition were digested by pancreatin and plated into a new well with 100 μL culture medium before detection. Cell proliferation was quantified on days 1, 3, and 7 by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) per the manufacturer’s instructions. The absorbance was measured at 450 nm using a Model 550 microplate reader (Bio-RAD, Hercules, California). Cell proliferation was expressed as a fold percentage of absorbance to the control level (day 0). The cell proliferation was expressed as a fold change of the absorbance of responding days vs day 0. Data were presented as mean±standard deviation. All experiments were performed in triplicate, and 3 independent repeated experiments were performed.
After 7 days of seeding marrow stem cells on the scaffold, the marrow stem cell–PLLGA composition was fixed immediately in 4 mL of 2.5% glutaraldehyde at 4°C for 1 day and then dehydrated with a graded series of 70%, 80%, 90%, and 100% ethanol. Critical-point drying was performed in liquid CO2 at 37°C. The specimens were vacuum-coated with a 5-nm layer of gold in a high-vacuum gold spatter coater and viewed with a JSM-5600LV SEM (JEOL, Tokyo, Japan).
Full-thickness Articular Cartilage Defect Repair
Twenty-seven Japanese white rabbits (54 knees) were anesthetized intravenously with 2.5% pentobarbital sodium at 1 mL/kg body weight. Full-thickness cylinder articular cartilage defects were created in the trochlea of the distal femur with a diameter of 4.5 mm and depth of 0.8 mm using a 4.5-mm-diameter corneal trephine. The knees were randomly divided into 3 groups: group 1 knees underwent microfracture with implanted cell-free PLLGA scaffolds; group 2 knees underwent microfracture; and group 3 knees underwent cell-free PLLGA scaffolds without microfracture. Microfracture was performed using a 25-gauge needle. The distance between pores was 1 mm with a depth to where only blood and lipid droplets came out. After microfracture in group 1, cell-free PLLGA scaffolds were filled into the defect, and incisions were closed in layers. After treatment, all rabbits were kept in cages freely with no immobilization. Every 3 rabbits were sacrificed by overdose anesthesia at weeks 6, 12, and 24 postoperatively.
Magnetic Resonance Imaging
The spoiled gradient recalled acquisition in steady state (SPGR) sequences (repetition time=60 ms, echo time=6 ms, flip angle=90°) were applied by a 3.0-T magnet scanner (Siemens, Berlin, Germany) with a small flex coil. Images of the repaired articular cartilage were taken of the sequences.
Histological Staining and Immunohistochemistry
The distal femurs were removed and fixed in 4% paraformaldehyde, decalcified in 10% ethylenediaminetetraacetic acid, and embedded in paraffin. Serial sections of 6 μm were cut horizontally along the maximum diameter of the repaired sites. Histological staining was performed with hematoxylin-eosin and toluidine blue, and immunohistochemistry staining was performed with anti-collagen type II monoclonal antibody at 1:200 dilution (Calbiochem, Darmstadt, Germany).
International Cartilage Repair Society Scores
International Cartilage Repair Society (ICRS) Cartilage Repair Assessment of Repaired Cartilage was used to evaluate cartilage regeneration under macroscopic outcomes, and the ICRS Visual Histological Assessment Scale of Repaired Cartilage was used to evaluate histological outcomes. The samples were evaluated by 2 observers blinded to the group identities (X. Zhang, J. Zhu). The results are expressed as box plots, which indicate median, lower, and upper quartiles. Spearman correlation was used to test the consistence of the scores by the 2 observers.
SPSS version 17.0 software (SPSS, Inc, Chicago, Illinois) was used for statistical analysis. The nonparametric test was used for the ICRS scores statistical analysis. The Kruskal-Wallis H test was used to compare the 3 groups, and the Mann-Whitney test was used between every 2 groups. The level of statistical significance was defined as P<.05.
Scanning electron microscopy revealed that the scaffolds were composed of 3-dimensional micron fibers. The fiber diameter was approximately 20 μm, and the pore size was 300 μm (Figure 1A). Scanning electron microscopy and phase contrast microscopy were also performed on day 7 after the cells were seeded on scaffolds and the marrow stem cells adhered to the scaffolds (Figures 1B, 1C). Marrow stem cell proliferation was assessed using the Cell Counting Kit-8. The fold changes were 1.65±0.47, 3.42±0.38, and 4.68±0.55 on days 1, 3, and 7, respectively (mean±standard deviation), indicating that marrow stem cells proliferated well on the scaffolds.
Figure 1: Characteristics and biocompatibility of poly(L-lactic-co-glycolic acid) (PLLGA) scaffolds. Scaffolds were observed by scanning electron microscopy (A). Rabbit marrow stem cells on the scaffold were observed by scanning electron microscopy (B) and phase contrast microscope (C). The proliferation of marrow stem cells on the PLLGA scaffold was examined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Data are expressed as mean±standard deviation from 3 experiments (D).
Magnetic resonance imaging demonstrated that the signal in group 1 was closer to normal articular cartilage than that in groups 2 and 3. In group 1, signals in some areas were lower than their surrounding areas even though the surface appeared smooth 6 weeks after implantation. However, at weeks 12 and 24 postoperatively, the signals became as homogeneous as the adjacent normal cartilage (Figures 2A–C). In group 2, the surface gradually became smoother; however, the signal remained heterogeneous at week 24 (Figures 2D–F). In group 3, the defect area showed a higher signal than the normal area at week 6 but became weaker at weeks 12 and 24 (Figures 2G–I).
Figure 2: Magnetic resonance imaging of rabbit knees after cartilage repair. In group 1 (microfracture with poly[L-lactic-co-glycolic acid] [PLLGA] implantation), some low intensity signals existed at week 6 (A) but disappeared at weeks 12 (B) and 24 (C). In group 2 (microfracture), low-intensity signals existed throughout the observation period (D–F). In group 3 (PLLGA implantation), the defect area showed higher signal than normal area at week 6 (G), but became weaker at weeks 12 (H) and 24 (I). Cartilage defect area (triangles).
The gross appearances showed that the regenerated tissue in group 1 was more similar to hyaline cartilage than those in groups 2 and 3. At week 6, the defect in group 1 was partially filled with white-semitransparent tissue, and the boundary between normal cartilage and regenerated tissue was distinct (Figure 3A). In group 2, a large vacant position still existed (Figure 3B). In group 3, the defect was covered by uneven white tissue in the center of the defect with a visible junction (Figure 3C). At week 12, the color and thickness of the regenerated tissue were similar to the surrounding normal tissue in group 1 (Figure 3D). In group 2, a thin layer of white and rough fiber-like tissue existed in the defect (Figure 3E). In group 3, fissures existed on the surface of newly formed tissue in the defect (Figure 3F). At week 24, the defect in group 1 was covered with semitransparent tissue, and the color and thickness were more similar to the adjacent normal cartilage with an indistinct boundary (Figure 3G). In group 2, the defect was covered with rough, fiber-like tissue with some fissures on the surface (Figure 3H). In group 3, the regenerative tissue and host cartilage were consecutive, but the boundary was distinct, despite similar thickness to the adjacent normal cartilage (Figure 3I). Microfracture combined with PLLGA scaffold improved cartilage regeneration.
Figure 3: Gross appearance of regenerated tissue. In group 1 (microfracture with poly(L-lactic-co-glycolic acid) [PLLGA] implantation), the defect could be identified at week 6 (A) but became smooth similar to the nearby normal cartilage at week 12 (B); hyaline-like cartilage existed at week 24 (C). In group 2 (microfracture), a large defect existed at week 6 (D) and was filled with white soft tissue at week 12 (E); the defect was covered with fiber-like cartilage tissue at week 24 (F). In group 3 (PLLGA implantation), the defect was covered by uneven white tissue at week 6 (G); a thin layer of white and rough, fiber-like tissue was formed at weeks 12 (H) and 24 (I).
Histological and immunohistochemical staining were also used for cartilage repair evaluation. Results showed that the 1-step repair method promoted cartilage regeneration.
At week 6, the boundary between normal cartilage and regenerated tissue in group 1 was distinct. Toluidine blue and collagen type II were stained positively (Figures 4a–c). In group 2, a large area defect existed on the trochlea covered with little tissue (Figures 4d–f). In group 3, the defect was filled with some fiber-like tissue, and the gap between the normal cartilage and regenerated tissue was apparent. Toluidine blue was positive, but collagen type II was stained weakly (Figures 4g–i).
Figure 4: Histological and immunohistochemistrical evaluations of regenerated tissue. Hematoxylin-eosin staining visualized the gross morphology (left panel), toluidine blue staining assessed glycosaminoglycans formation (middle panel), and collagen type II staining identified chondrocytes (right panel). Boundary between normal and regenerated tissue was clear at week 6 in groups 1 (a–c), 2 (d–f), and 3 (g–i). At week 12, in group 1, chondrocytes and cartilage lacuna existed. Junction gap was partially integrated (j–l). In group 2, the defect was covered with granulation-like tissue, and the cells were disorderly (m–o). In group 3, fiber-like regenerated tissue filled the defect, and the tide line was not as smooth as normal cartilage (p–r). At week 24, in group 1, cells were arranged as normal cartilage with less extracellular matrix than normal cartilage (s–u). It was mostly integrated with surrounding cartilage. In group 2, the defect was all covered with rough fiber-like tissue with some chondrocytes in the extracellular matrix (v–x). In group 3, chondrocytes and cartilage lacuna could not be seen in regenerated tissue, and collagen type II stained slightly positive (y–z’). Border of normal cartilage and regenerated tissue, with normal cartilage on the left and regenerated tissue on the right (dotted lines).
At week 12, the regenerated tissue thickness was similar to the surrounding normal tissue in group 1. Chondrocytes and cartilage lacuna existed. Junction gaps existed between the regenerated and host cartilage (Figures 4j–l). In group 2, the defect was covered with granulation-like tissue, with cells arranged disorderly and no cartilage lacuna. Toluidine blue was slightly positive (Figures 4m–o). In group 3, fiber-like regenerated tissue filled the entire defect, and the tide line was not as smooth as normal cartilage. The junction gap was not as distinct as it was at week 6 (Figures 4p–r).
At week 24, the defect was filled with hyaline-like cartilage tissue in Group 1. Collagen type II and toluidine blue were stained positively as normal cartilage. Although less extracellular matrix existed than in normal cartilage, the cells were regularly arranged. The repaired tissue was mostly integrated with surrounding cartilage. The defect was covered with semitransparent tissue, and the color and thickness were more similar to the adjacent normal cartilage than in groups 2 and 3. The boundary was indistinct (Figures 4s–u). In group 2, the defect was covered with rough, fiber-like tissue with some fissures on the surface. The boundary was distinct with a similar thickness to the adjacent cartilage (Figures 4v–x). In group 3, no chondrocytes or cartilage lacuna existed in regenerated tissue, and collagen type II did not stain as positively as did group 1 (Figures 4y–z′).
These results demonstrated that microfracture combined with PLLGA scaffold could stimulate articular cartilage regeneration more rapidly and effectively than microfracture.
International Cartilage Repair Society macroscopic scores (r2=0.700, P<.001) and ICRS histological scores (r2=0.739, P<.001) by the 2 observers (X. Zhang, J. Zhu) were consistently tested by Spearman correlation. International Cartilage Repair Society scores after repair confirmed that microfracture combined with PLLGA promoted cartilage regeneration for macroscopic and microscopic assessments (Figures 5A, B). Scores increased as time passed in all groups. Group 1’s scores were significantly higher than those of groups 2 and 3 at each time point ( , P<.05; , P<.01).
Figure 5: International Cartilage Repair Society (ICRS) scores of regenerated tissue. The ICRS Cartilage Repair Assessment of Repaired Cartilage (macroscopic outcomes) (A). Scores of the ICRS Visual Histological Assessment Scale of Repaired Cartilage (histological outcomes) (B). Group 1, microfracture with poly(L-lactic-co-glycolic acid) implantation; group 2, microfracture; group 3, poly(L-lactic-co-glycolic acid) implantation. Group 1’s scores were significantly higher than those of groups 2 and 3 at each time point ( , P<.05, , P<.01).
In the current study, microfracture with cell-free PLLGA scaffold implantation (group 1) provided faster and more effective cartilage regeneration than microfracture (group 2) or cell-free PLLGA implantation (group 3). Although microfracture regenerates fiber-like cartilage and cell-free scaffold implanting regenerates fiber or hyaline-like cartilage, the repairing effect and speed were unsatisfactory. The authors’ 1-step cartilage repair, in which microfracture was combined with cell-free PLLGA scaffold implantation, effectively promoted hyaline-like cartilage regeneration in adult animals with large-area articular cartilage defects. Moreover, this method is easier to perform than other cartilage tissue engineering. The techniques and materials are commonly used in clinical applications. Microfracture is considered a first-line technique to treat small cartilage defects. Poly(L-lactic-co-glycolic acid) scaffold has been approved by the Food and Drug Administration for clinical applications.16 Although neither technique is new, their combination is novel. This 1-step cartilage repair method could be used clinically under arthroscopy.
The PLLGA scaffold supported marrow stem cell growth in vitro. The traditional tissue engineering procedure (ie, harvesting and expanding marrow stem cells and reimplanting) was not followed in vivo. A microenvironment for autologous marrow stem cells homing to the defect site was used in combination with cell-free PLLGA scaffold implantation to repair the defect to avoid a second anesthesia and surgery, which increase the risk of infection. The ideal regeneration results were obtained in vivo.
The stem cell niche is a microenvironment for stem cell self-renewal, proliferation, differentiation, mobilization, and homing.13,14,17 Microfracture produced an environment for endogenous stem cell homing instead of exogenous stem cell transplantation. An appropriate microenvironment is critical for cartilage regeneration in vivo.18 In the current study, microfracture was the key procedure to provide a suitable microenvironment. First, besides marrow stem cells, other important factors could be released through the microfracture channel, such as platelet-derived growth factor, transforming growth factor-β, bone morphogenetic proteins, and fibroblast growth factor, which induce marrow stem cells to differentiate into chondrocytes.19–22 Second, recent research indicated that platelet-rich plasma promotes the differentiation of marrow stem cells into chondrocytes.23 Third, crosstalk between extracellular matrix components of the microenvironment and marrow stem cells in the cartilage could further contribute to the differentiation of marrow stem cells into chondrocytes.18
Poly(L-lactic-co-glycolic acid) is the most commonly used absorbable implant in orthopedics.24 As a type of synthetic polymers, PLLGA has hypotoxicity and low immunogenicity compared with other protein products, such as collagen.25 Thus, we selected PLLGA for the cartilage scaffolds. In the current study, PLLGA scaffolds were 3-dimensional and composed of a fiber structure. The 3-dimensional structure with proper pore sizes provided desirable spaces for cell living and growth. Microfracture allowed blood and bone marrow to effuse into the scaffolds and allowed platelet and blood coagulation factors to induce blood clot formation in the scaffolds. Furthermore, the scaffolds could act as temporary mechanical supports for joint movement during regeneration due to their stiffness and elasticity to some degree.26
The implantation of marrow stem cells or chondrocytes expanded in culture in vitro is difficult to perform, and the risk exists of infection. When PLLGA scaffolds were combined with microfracture, their advantages strengthened, and their disadvantages diminished. Scaffolds caught and held marrow stem cells, cytokine, and blood in their spaces, providing temporary mechanical support and an appropriate microenvironment for marrow stem cells to renew, mobilize, and differentiate into chondrocytes.20 Marrow stem cells were contained in the repaired site instead of diffusing into an articular cavity. Consequently, their cartilage regeneration effect was maximized.
Although the long-term effects of the PLLGA scaffolds and microfracture combination in cartilage repair has not been observed, the advantage of this method is evident. The procedure is simple and effective, indicating clinical applicability.
Marrow stem cells adhere to and proliferate in the PLLGA scaffold. Compared with implanting the cell-free PLLGA scaffold or the microfracture alone, the combination of microfracture and cell-free PLLGA scaffolds uses endogenous marrow stem cells in situ and promotes cartilage regeneration rapidly and effectively in vivo microenvironments. Stem cell niches play a significant role in the cartilage repair process. The rabbit model provides the experimental basis for a 1-step cartilage repair method in which patients would be spared second anesthesia and surgery, decreasing the risk of infection and increasing clinical applicability.
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