A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

Purpose

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide^®) scaffold.

Methods

By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water–dioxane–PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT–PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide^® scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O’Driscoll score.

Results

In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide^® scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide^® scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells.

Conclusion

The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide^® scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide^® scaffold with cells.     

不同细胞移植修复兔全层关节软骨缺损的比较研究

目的 :比较软骨细胞、骨髓基质细胞及成纤维细胞对全层关节软骨缺损的修复作用。材料和方法 :取幼兔的软骨细胞、骨髓基质细胞及成纤维细胞 ,共 3种有生成软骨潜力的细胞进行体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 8只成年新西兰大白兔的股骨滑车关节面上造成直径 4 5mm、深 3 0mm的全层关节软骨缺损 ,将 3种细胞 -PLA复合物分别植入关节软骨缺损处。植入细胞 -PLA复合物为实验组 ,单纯植入PLA支架为对照组。术后 6周、12周观察缺损修复情况及新生组织类型。结果 :软骨细胞移植组为软骨样组织修复 ,分界明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨部分重建 ;细胞排列紊乱。骨髓基质细胞移植组为软骨样组织修复 ,分界不明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨重建良好 ,软骨下潮线恢复 ;细胞排列趋于正常。成纤维细胞移植组为纤维组织修复 ,甲苯胺兰及Ⅱ型胶原染色阴性 ;软骨下潮线消失。对照组为纤维组织修复。结论 :软骨细胞、骨髓基质细胞移植修复软骨缺损明显优于成纤维细胞及对照组。骨髓基质细胞与软骨细胞移植组的修复结果无统计学差异 ,但骨髓基质细胞修复组织的细胞排列有序 ,软骨下骨重建良好 ,与周围组织融合密切 ,更接近正?     

In vitro bonding of pre-seeded chondrocytes

Abstract This study investigated the capacity of seeded chondrocytes to join separate cartilage disc matrices in an in vitro model. Articular cartilage discs were harvested from pigs and devitalized by multiple freeze/thaw cycles. The devitalized cartilage discs were incubated in the presence (experimental group) or absence (control group) of chondrocytes for 10 days in order to allow chondrocytes to adhere to the matrix. After culturing, pairs of cartilage discs were held in apposition in a 48-multiwell plate and cultured for two and eight weeks. Twelve experimental composites (with cells) and twelve controls (without cells) were prepared per each time point. Samples were retrieved from culture and grossly inspected for adherence and processed for histological evaluation. Histological sections demonstrated the presence of new cartilage matrix formed by seeded chondrocytes bonding the two matrix discs together and producing glycosaminoglycans (GAG) able to diffuse within the devitalized tissue. Generally, gross adherence between the discs was demonstrated in the experimental samples, while the controls did not show any bonding. We conclude that isolated and seeded chondrocytes produce a new cartilaginous matrix, capable to join devitalized cartilage discs in vitro.     

A comparative study of 3 different cartilage repair techniques

Purpose  

The value of cell-free techniques in the treatment of cartilage defects remains under debate. In this study, cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with a cell-free collagen type-I gel or a collagen type-I gel seeded with autologous chondrocytes. As a control, abrasion arthroplasty was included.     

Effect of blood on the morphological,biochemical and biomechanical properties of engineered cartilage

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days’ contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the “long-term effect” of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.     

Early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle

Matrix-induced autologous chondrocyte implantation (MACI) is a tissue-engineering technique for the treatment of full-thickness articular cartilage defects and requires the use of a three-dimensional collagen type I–III membrane seeded with cultured autologous chondrocytes. The cell-scaffold construct is implanted in the debrided cartilage defect and fixed only with fibrin glue, with no periosteal cover or further surgical fixation. In a clinical pilot study, the MACI technique was used for the treatment of full-thickness, weight-bearing chondral defects of the femoral condyle in 16 patients. All patients were followed prospectively and the early postoperative attachment rate, 34.7 days (range: 22–47) after the scaffold implantation, was determined. With the use of high-resolution magnetic resonance imaging (MRI), the transplant was graded as completely attached, partially attached, or detached. In 14 of 16 patients (87.5%), a completely-attached graft was found, and the cartilage defect site was totally covered by the implanted scaffold and repair tissue. In one patient (6.25%), a partial attachment occurred with partial filling of the chondral defect. A complete detachment of the graft was found in one patient (6.25%), which resulted in an empty defect site with exposure of the subchondral bone. Interobserver variability for the MRI grading of the transplants showed substantial agreement (=0.775) and perfect agreement (_w=0.99). In conclusion, the implantation and fixation of a cell-scaffold construct in a deep cartilage defect of the femoral condyle with fibrin glue and with no further surgical fixation leads to a high attachment rate 34.7 days after the implantation, as determined with high resolution MRI.     

Autologous chondrocyte implantation: cells phenotype and proliferation analysis

The phenotype and proliferation of human chondrocytes in culture were analyzed before they were implanted as autologous graft in cartilage lesions. During ten autologous chondrocyte implantations in articular cartilage lesions of the knee in ten patients, small amounts of cells to be implanted were collected and analyzed by morphology, cytochemistry (alcian blue, safranin-O), and immunocytochemistry (antibodies anti-S100 protein, anti-collagen types I and II, anti-chondroitin-S). In four cases the cells were also cultured for 3 weeks. At 1, 10, and 20 days of culture cells were incubated with 1 microCi/ml [3H]thymidine for proliferation analysis. In all cases the cells showed the morphological appearance of mature chondrocytes, stained positively for alcian blue and safranin-O, and revealed a strong immunoreaction for S-100 protein, type II collagen, and chondroitin-S but not for type I collagen. Radioisotope assay of chondrocyte proliferation at 1, 10, and 20 days of culture revealed a progressive increase in [3H]thymidine incorporation. These findings indicate that the cells before autologous implantation maintain their differentiated phenotype of mature chondrocytes and proliferate greatly. This analysis is an essential step preceding wider use of this treatment in humans. However, other biological aspects of the autologous chondrocyte graft remain to be elucidated.     

软骨细胞在微载体中的培养和快速扩增

目的：探索在短期内获得大量成活率高、分化良好的兔关节软骨细胞的方法,方法：应用胰蛋白酶、胶酶消化的方法从新生新西兰兔关节软骨处分离、培养软骨细胞,并将获得的软骨细胞在旋转生物反应应（RCCS）内应用Cytodex-3微载体进行培养。应用倒置显微镜和扫描电镜对微载体表面的软骨细胞进行动态观察,并对收获的软骨细胞进行Ⅰ、Ⅱ型胶原的细胞免疫化学染色分析。结果：并节软骨细胞可快速贴附于Cytodex-3微载体表面,细胞伸展后生长加速,到培养后期,细胞密度可达最初接种的20倍。在微载体上收获的软骨细胞Ⅰ型胶原的免疫细胞化学染色呈阴性,Ⅱ型胶原染色则呈强阳性。结论：微载体细胞培养技术是一种简便、快速的体外细胞扩增方法,可为构体建组织工程化人工软骨提供大量软骨软件。     

Effect of in vitro culture on a chondrocyte-fibrin glue hydrogel for cartilage repair

Research in tissue engineering has been focused on articular cartilage repair for more than a decade. Some pioneristic studies involved the use of hydrogels such as alginate and fibrin glue which still possess valuable potential for cartilage regeneration. One of the main issues in cartilage tissue engineering is represented by the ideal maturation of the construct, before in vivo implantation, in order to optimize matrix quality and integration. The present study was focused on the effect of in vitro culture on a fibrin glue hydrogel embedding swine chondrocytes. We performed an evaluation of the immunohistochemical and biochemical composition and of the biomechanical properties of the construct after 1 and 5 weeks of culture. We noticed that chondrocytes survived in the fibrin glue gel and enhanced their synthetic activity. In fact, DNA content remained stable, while all indices of cartilage matrix production increased (GAGs content, immunohistochemistry for collagen II and safranin-o staining). On the other hand, the biomechanical properties remained steady, indicating a gradual substitution of the hydrogel scaffold by cartilaginous matrix. This demonstrates that an optimal preculture could provide the surgeon with a better engineered cartilage for implantation. However, whether this more mature tissue will result in a more efficient regeneration of the articular surface still has to be evaluated in future investigations.     

关节软骨表层组织的免疫防护作用

文中的实验采用免疫荧光组织学方法观察到正常的关节软骨表层组织可阻止抗Ⅱ型胶原抗体侵入软骨深部组织,防止抗体与软骨细胞结合,具有免疫防护作用。     

雌激素对软骨细胞胶原表型表达的影响

目的 :观察雌激素对体外培养兔关节软骨细胞胶原表型表达的影响。方法 :体外培养雌兔关节软骨细胞 ,随机分为A、B两组 ,A组中加入 1 7β -雌二醇 0mol/L、1 0 - 6 mol/L、1 0 - 7mol/L、1 0 - 8mol/L、1 0 - 9mol/L、1 0 - 10 mol/L、1 0 - 11mol/L、1 0 - 12 mol/L干预 72小时 ;B组先用 1 0ng/mlIL - 1 β干预 2 4小时 ,随后分别加入 0mol/L、1 0 - 6 mol/L～ 1 0 - 12 mol/L 1 7β -雌二醇作用 72小时。采用RT-PCR方法观察软骨细胞Ⅰ、Ⅱ、Ⅲ型胶原表达。结果 :1 0 - 6 mol/L雌二醇或单用IL - 1 β均抑制正常软骨细胞Ⅱ型胶原mRNA表达 ,低浓度雌二醇 (1 0 - 11、1 0 - 12 mol/L)能够对抗IL - 1 β的抑制作用 ;所有软骨细胞均未表达Ⅲ型胶原mRNA ;几乎所有浓度雌二醇 (1 0 - 12 mol/L除外 )均诱导软骨细胞表达Ⅰ型胶原。结论 :雌激素对关节软骨细胞胶原表型表达的调控作用随其浓度变化而不同。对变性软骨细胞而言 ,低于生理浓度的雌激素 (1 0 - 12 mol/L)对维持其胶原表型最适宜。     

Treatment of articular cartilage lesions of the knee joint using a modified AMIC technique

This study describes a modified AMIC technique consisting of perforations according to Pridie, rather than microfractures, and the covering of the focus of the lesion with a biological collagen patch enriched with bone marrow blood drawn through the knee itself. This technique allows advantages of both the Pridie technique and the in situ proliferation of mesenchymal cells beneath a biological collagen membrane, ‘augmented’, with bone marrow blood. The collagen membrane forms the roof of a ‘biological chamber’, and serves to protect and contains the stem cells as they differentiate into chondrocytes, which will form a healthy regenerative cartilage.     

ACL reconstruction in a rabbit model using irradiated Achilles allograft seeded with mesenchymal stem cells or PDGF-B gene-transfected mesenchymal stem cells

The present study was conducted to develop a new strategy to accelerate reconstruction of the anterior cruciate ligament (ACL) by modifying the Achilles allograft with autogenous mesenchymal stem cells (MSCs) or PDGF-B transfected MSCs in a rabbit model. The allografts were first irradiated with Co⁶⁰, stored at −80°C, and then seeded with cells for implantation. Bilateral ACL reconstructions were performed. On the left, the allograft was either seeded with MSCs or PDGF-B transfected MSCs and acted as the experimental group. On the right, the graft without any cells seeded acted as control. At 3, 6 and 12 weeks after surgery, histological observation found that implatation of MSCs or PDGF-B transfected MSCs accelerated cellular infiltration into the ACL and enhanced collagen deposition in the wound. PDGF-B transfected MSCs could also lead to an initial promotion of angiogenesis. This gene transfer technique or cell implantion may be a potentially useful tool for improving ligament remodeling.     

Increased chondrocyte seeding density has no positive effect on cartilage repair in an MPEG-PLGA scaffold

Purpose

This study investigates the effect of cell seeding density on cartilage repair in matrix-assisted chondrocyte implantation in vitro and in vivo.

Methods

In vitro: Four different cell seeding densities of human chondrocytes were seeded onto a porous methoxy-polyethylene glycol-polylactic-co-glycolic acid scaffold (MPEG-PLGA) polymer scaffold ASEED? (1.2 × 10⁶, 4.0 × 10⁶, 1.2 × 10⁷ and 2.0 × 10⁷ cells/cm³). The cartilage repair response was evaluated by relative gene expression of the chondrogenic markers sox9, collagen types I, II and X, and aggrecan, total DNA content and sulphated glycosaminoglycan synthesis. In vivo: Using a New Zealand white rabbit intercondylar osteochondral defect model, three different cell seeding densities (1.2 × 10⁶, 4.0 × 10⁶ and 1.2 × 10⁷ cells/cm³) were tested with an empty scaffold as control. The cartilage repair response was evaluated using O’Driscoll score.

Results

In vitro: A significant difference (p < 0.05) in total DNA content was found at day 2 but not at day 7. The low cell seeding densities yielded the highest GAG content (p < 0.001) at day 7. Collagen type I was highest (p < 0.01) at the lowest density at day 7. In vivo: No significant difference was found between the 4 groups.

Conclusions

No positive effect on cartilage repair was found using increased cell seeding density.

Level of evidence

Controlled experimental study, Level II.     

骨形态发生蛋白对培养胎儿关节软骨细胞代谢的影响及意义

为了解骨形态发生蛋白（ＢＭＰ）在关节软骨损伤与修复及异位化骨中的作用，本研究观察了部分纯化的牛骨形态发生蛋白（ｂＢＭＰ）和高度纯化的人骨形态发生蛋白（ｈＢＭＰ）对体外贴壁培养原代、反分化人胎儿关节软骨细胞及鼠３Ｔ３成纤维细胞ＤＮＡ、胶原和蛋白多糖合成的影响。结果发现：部分纯化的ｂＢＭＰ和高度纯化的ｈＢＭＰ均不促进原代人关节软骨细胞合成ＤＮＡ和胶原，并显著抑制其蛋白多糖的合成。部分纯化的ｂＢＭＰ对反分化关节软骨细胞和鼠３Ｔ３成纤维细胞的ＤＮＡ合成、胶原合成和蛋白多糖合成却有明显促进作用。因此，本研究认为，ＢＭＰ诱导下的关节软骨细胞不会发生反分化，而有可能进一步向肥大软骨细胞样细胞或成骨细胞样细胞方向分化，并可能使反分化的软骨细胞重新表达软骨细胞表型。     

A novel implantation technique for engineered osteo-chondral grafts

We present a novel method to support precise insertion of engineered osteochondral grafts by pulling from the bone layer, thereby minimizing iatrogenic damage associated with direct manipulation of the cartilage layer. Grafts were generated by culturing human expanded chondrocytes on Hyaff^®-11 meshes, sutured to Tutobone^® spongiosa cylinders. Through the bone layer, shaped to imitate the surface-contours of the talar dome, two sutures were applied: the first for anterograde implantation, to pull the graft into the defect, and the second for retrograde correction, in case of a too deep insertion. All grafts could be correctly positioned into osteochondral lesions created in cadaveric ankle joints with good fit to the surrounding cartilage. Implants withstood short-term dynamic stability tests applied to the ankle joint, without delamination or macroscopic damage. The developed technique, by allowing precise and stable positioning of osteochondral grafts without iatrogenic cartilage damage, is essential for the implantation of engineered tissues, where the cartilage layer is not fully mechanically developed, and could be considered also for conventional autologous osteochondral transplantation.     

Autologous collagen-induced chondrogenesis technique (ACIC) for the treatment of chondral lesions of the talus

Purpose

Autologous collagen-induced chondrogenesis technique (ACIC) combines microfractures with the use of an injectable atelocollagen matrix that allows performing the whole cartilage repair treatment arthroscopically. The aim of this study was to evaluate the in vitro cytocompatibility of this biomaterial using human bone marrow mesenchymal stem cells and human chondrocytes. Moreover, the preliminary data of five patients affected by chondral lesion of the talus treated with the ACIC technique are shown.

Methods

Human bone marrow mesenchymal stem cells and human chondrocytes were seeded on solid and pre-solid atelocollagen scaffolds. Cell–scaffold constructs were cultured for 7 days and then prepared for histological analyses. Arthroscopic ACIC was performed in five patients affected by chondral lesions of the talus; they were clinically evaluated with AOFAS, VAS and Tegner score before and then after 6 months from surgery.

Results

In vitro results showed that both bone marrow mesenchymal stem cells and chondrocytes were able to efficiently colonize the whole construct, from the surface to the core, only when seeded on the pre-solid atelocollagen scaffold, but not on its solid form. No adverse events were observed in the patients treated with the ACIC technique; a significant improvement in VAS pain scale and in AOFAS score was found at 6 months follow up.

Conclusion

Injectable atelocollagen can be considered a feasible scaffold for cartilage repair treatment, in particular if used in its pre-solid form. ACIC leads to good clinical results in the treatment for chondral lesions of the talus even if longer follow-up and a higher number of patients are necessary to confirm these data.

Level of evidence

IV.     

同种异体软骨细胞/聚乳酸复合物即时修复兔耳廓软骨缺损

 目的观察用同种异体软骨细胞/聚乳酸(Poly-DL-lactide,PDLLA)复合物在体内即时修复软骨缺损的能力.方法将兔耳廓软骨细胞体外分离消化,以PDLLA为支架,用软骨细胞/PDLLA复合物即时移植修复兔耳廓软骨缺损,对照组采用PDLLA,6、12、18周后观察软骨缺损修复情况.结果实验组移植后18周,软骨缺损愈合,修复软骨厚度均匀.对照组缺损区为条索状纤维组织修复,中央部凹陷.结论同种异体软骨细胞/PDLLA复合物即时修复软骨缺损是一种非常有前途的且适合临床应用的组织工程学方法.     

Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo

Purpose

The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested.

Methods

Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo.

Results

After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants.

Conclusion

The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.     

Chondrogenesis in a hyaluronic acid scaffold: comparison between chondrocytes and MSC from bone marrow and adipose tissue

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.