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.     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

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.     

聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损

目的：应用聚乙醇酸（ＰＧＡ）负载的兔软骨细胞培养移植修复同种异体关节软骨缺损．方法：应用在生物体内可降解吸收、纤维状多孔态的ＰＧＡ作为支架行兔软骨细胞培养．培养１４天后，软骨细胞在ＰＧＡ提供的三维空间中大量分裂、增殖并合成大量软骨基质，形成ＰＧＡ－软骨细胞复合体，然后利用该复合体移植修复同种异体兔膝关节全层软骨缺损，对侧膝关节作对照．术后行大体、组织学、电镜动态观察及修复组织厚度测定．结果：ＰＧＡ在术后８周完全降解吸收，实验侧与对照侧修复组织的厚度有显著性差异（Ｐ＜０．０１）；术后１６周在实验侧可见典型的软骨组织，电镜下为成熟的软骨细胞，而对照侧为纤维组织修复．结论：应用ＰＧＡ－软骨细胞复合体移植，可修复同种异体的兔关节软骨缺损，为临床治疗关节软骨缺损奠定了基础．     

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.     

Biochemical and biomechanical properties of lesion and adjacent articular cartilage after chondral defect repair in an equine model

BACKGROUND: Chondral defects may lead to degradative changes in the surrounding cartilage, predisposing patients to developing osteoarthritis. PURPOSE: To quantify changes in the biomechanical and biochemical properties of the articular cartilage adjacent to chondral defects after experimental defect repair. STUDY DESIGN: Controlled laboratory study. METHODS: Specimens were harvested from tissue within (lesion), immediately adjacent to, and at a distance from (remote area) a full-thickness cartilage defect 8 months after cartilage repair with genetically modified chondrocytes expressing insulin-like growth factor-I or unmodified, control chondrocytes. Biomechanical properties, including instantaneous Young's and equilibrium aggregate moduli, were determined by confined compression testing. Biochemical properties, such as water and proteoglycan content, were also measured. RESULTS: The instantaneous Young's modulus, equilibrium modulus, and proteoglycan content increased, whereas water content decreased with increasing distance from the repaired lesion. The instantaneous Young's and equilibrium moduli of the adjacent articular cartilage were 80% and 50% that of remote area samples, respectively, whereas water content increased 0.9% and proteoglycan content was decreased by 35%. No significant changes in biomechanical and biochemical properties were found either in the lesion tissue or in adjacent cartilage with genetic modification of the chondrocytes. CONCLUSION: Articular cartilage adjacent to repaired chondral defects showed significant remodeling 8 months after chondral defect repair, regardless of whether genetically modified or unmodified cells were implanted. CLINICAL RELEVANCE: Changes in the biochemical and biomechanical properties of articular cartilage adjacent to repaired chondral defects may represent remodeling as part of an adaptive process or degeneration secondary to an altered distribution of joint forces. Quantification of these changes could provide important parameters for assessing progress after operative chondral defect repair.     

Cell-based therapy for meniscal repair: a large animal study

BACKGROUND: The avascular portion of the meniscus cartilage in the knee does not have the ability to repair spontaneously. HYPOTHESIS: Cell-based therapy is able to repair a lesion in the swine meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Sixteen Yorkshire pigs were divided into four groups. A longitudinal tear was produced in the avascular portion of the left medial meniscus of 4 pigs. Autologous chondrocytes were seeded onto devitalized allogenic meniscal slices and were secured inside the lesion with two sutures. Identical incisions were created in 12 other pigs, which were used as three separate control groups: 4 animals treated with an unseeded scaffold, 4 were simply sutured, and 4 were left untreated. Meniscal samples were collected after 9 weeks, and the samples were analyzed grossly, histologically, and histomorphometrically. RESULTS: Gross results showed bonding of the lesion margins in the specimens of the experimental group, whereas no repair was noted in any of the control group specimens. Histological and histomorphometrical analysis showed multiple areas of healing in the specimens of the experimental group. CONCLUSIONS: This study demonstrated the ability of seeded chondrocytes to heal a meniscal tear. Clinical Relevance: Cell-based therapy could be a potential tool for avascular meniscus repair.     

Blood exposure has a negative effect on engineered cartilage

Purpose  

The aim of this study was to investigate the in vitro effect of different concentrations of blood on the morphological and biochemical properties of engineered cartilage. Previous studies have demonstrated a negative effect of blood on native cartilage; however, the effect of the contact of blood on engineered cartilage is unclear.     

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。     

An allogenic cell-based implant for meniscal lesions

BACKGROUND: Meniscal tears in the avascular zones do not heal. Although tissue-engineering approaches using cells seeded onto scaffolds could expand the indication for meniscal repair, harvesting autologous cells could cause additional trauma to the patient. Allogenic cells, however, could provide an unlimited amount of cells. HYPOTHESIS: Allogenic cells from 2 anatomical sources can repair lesions in the avascular region of the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Both autologous and allogenic chondrocytes were seeded onto a Vicryl mesh scaffold and sutured into a bucket-handle lesion created in the medial menisci of 17 swine. Controls consisted of 3 swine knees treated with unseeded implants and controls from a previous experiment in which 4 swine were treated with suture only and 4 with no treatment. Menisci were harvested after 12 weeks and evaluated histologically for new tissue and percentage of interface healing surface; they were also evaluated statistically. RESULTS: The lesions were closed in 15 of 17 menisci. None of the control samples demonstrated healing. Histologic analysis of sequential cuts through the lesion showed formation of new scar-like tissue in all experimental samples. One of 8 menisci was completely healed in the allogenic group and 2 of 9 in the autologous group; the remaining samples were partially healed in both groups. No statistically significant differences in the percentage of healing were observed between the autologous and allogenic cell-based implants. CONCLUSION: Use of autologous and allogenic chondrocytes delivered via a biodegradable mesh enhanced healing of avascular meniscal lesions. CLINICAL RELEVANCE: This study demonstrates the potential of a tissue-engineered cellular repair of the meniscus using autologous and allogenic chondrocytes.     

A tissue engineered osteochondral plug: an in vitro morphological evaluation

Articular cartilage lesions have a poor intrinsic healing potential. The repair tissue is often fibrous, having insufficient biomechanical properties, which could frequently lead to the development of early osteoarthritis. In the last decade, tissue engineering approaches addressed this topic in order to restore joint function with a differentiated and functional tissue. Many biomaterials and techniques have been proposed and some of them applied in clinical practice, even though several concerns have been raised on the quality of the engineered tissue and on its integration in the host joint. In this study, we focused on engineering in vitro a biphasic composite made of cellular fibrin glue and a calcium–phosphate scaffold. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to allow a more efficient integration of the engineered tissue with the host. However, a firm in vitro bonding between the two components of the composite is a necessary condition to validate this model. Our study demonstrated a gross and microscopic integration of the two components and a cartilage-like quality of the newly formed matrix. Moreover, we noticed an improvement of this integration and GAGs production during the in vitro culture.     

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.     

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?: a morphological and molecular study of OCD fragments

Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in “bundles” and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae close to mineralized “islands” in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co‐localization within the islands of types I, II, X collagens and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non‐conclusive, only single nucleotide polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification.     

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.     

Histological changes and apoptosis of cartilage layer in human anterior cruciate ligament tibial insertion after rupture

The purpose of this study is to investigate the histological changes and apoptosis of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertion at different time periods after rupture. By using a core reamer, 35 tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions (number of days after injury: 19–206 days). A histological examination was performed and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect apoptosis. The average thickness of the cartilage layer, the glycosaminoglycan-stained area and the number of chondrocytes per millimeter decreased with time. The percentage average of TUNEL-positive chondrocytes was 42.0 ± 16.2. The histological degenerative changes of the cartilage layer in the ruptured ACL tibial insertion progressed with time, especially in the first 2 months. Moreover, chondrocyte apoptosis continued from 19 to 206 days after rupture. The results may help elucidate the etiology of the histological changes of the insertion, and may help in devising optimal treatment protocols for ACL injuries if apoptosis is controlled. Moreover, we consider that using a surviving ligament and minimizing a debridement of ACL remnant during ACL reconstruction may be important for ACL reconstruction to maintain cartilage layers in ACL insertions.     

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.     

Adhesion and collagen production of human tenocytes seeded on degradable poly(urethane urea)

Purpose

The aim of this study was to investigate whether human tenocytes taken from ruptured quadriceps tendon could be seeded on a biodegradable polycaprolactone-based polyurethanes (PU) urea scaffold. Scaffold colonization and collagen production after different culture periods were analyzed to understand whether tenocytes from ruptured tendons are able to colonize these biodegradable scaffolds.

Methods

Human primary tenocyte cultures of ruptured quadriceps tendons were seeded on PU scaffolds. After 3, 10 and 15 days of incubation, the samples were stained with haematoxylin and eosin and were examined under white light microscopy. After 15 and 30 days of incubation, samples were examined under transmission electron microscope. Total collagen accumulation was also evaluated after 15, 30 and 45 days of culture.

Results

After 15 and 30 days of culture, tenocyte-seeded scaffolds showed cell colonization and cell accumulation around interconnecting micropores. Tenocyte phenotype was variable. Collagen accumulation in seeded scaffolds demonstrated a progressive increase after 15, 30 and 45 days of culture, while control non-seeded scaffolds show no collagen accumulation.

Conclusion

These results showed that human tenocytes from ruptured quadriceps tendon can be seeded on polycaprolactone-based PU urea scaffolds and cultured for a long time period (45 days). This study also showed that human tenocytes from ruptured tendons seeded on PU scaffolds are able to penetrate the scaffold showing a progressively higher collagen accumulation after 15, 30 and 45 days of incubation. This study provides the basis to use this PU biodegradable scaffold in vivo as an augmentation for chronic tendon ruptures and in vitro as a scaffold for tissue engineering construct.     

关节软骨细胞体外培养时生物学性状的变化

<正> 在运动医学领域中,由于运动员的关节软骨伤病发生率远高于普通人,同时又缺乏有效的治疗方法,影响训练和运动成绩的提高,因此对关节软骨伤病的研究一直是重点科研项目之一。对关节软骨进行研究的手段近20年来有了巨大进步,其标志是关节软骨细胞在体外分离培养取得成功。它作为探索软骨细胞性状的先进手段正越来越广泛地被应用到关节软骨损伤和疾患的研究工作中。我所1982年在体外分离培养关节软骨细胞获得成功。近年来,对关节软骨细胞在体外培养过程中所     

Patellofemoral full-thickness chondral defects treated with Hyalograft-C: a clinical, arthroscopic, and histologic review

BACKGROUND: Tissue engineering has emerged as a potential therapeutic option for cartilage regeneration. HYPOTHESIS: Hyaluronan-based scaffolds seeded with autologous chondrocytes are a viable treatment for damaged articular surface of the patellofemoral joint. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Thirty-two chondral lesions with a mean size of 4.7 cm(2) were treated with Hyalograft-C. Twenty-two lesions were located in the patella and 10 in the trochlea. Sixteen patients had previous trauma, 3 had osteochondritis dissecans, and 13 had degenerative changes. Transplantations were carried out arthroscopically or through a miniarthrotomy incision. Eight patients had concomitant procedures, including patellar realignment (2), lateral release (3), and meniscectomy (3). Results were evaluated using the International Cartilage Repair Society-International Knee Documentation Committee scale, EuroQol EQ-5D form, and magnetic resonance imaging scans at 12 and 24 months. Six patients had second-look arthroscopy and biopsies. Statistical analysis was performed using the paired t test and Wilcoxon signed rank test. RESULTS: The International Cartilage Repair Society-International Knee Documentation Committee and EuroQol EQ-5D scores demonstrated a statistically significant improvement (P < .0001). Objective preoperative data improved from 6/32 (18.8%) with International Knee Documentation Committee A or B to 29/32 (90.7%) at 24 months after transplantation. Mean subjective scores improved from 43.2 points preoperatively to 73.6 points 24 months after implantation. Magnetic resonance imaging studies at 24 months revealed 71% to have an almost normal cartilage with positive correlation to clinical outcomes. Second-look arthroscopies in 6 cases revealed the repaired surface to be nearly normal with biopsy samples characterized as hyaline-like in appearance. CONCLUSION: Biodegradable scaffolds seeded with autologous chondrocytes can be a viable treatment for chondral lesions. The type of tissue repair achieved demonstrated histologic characteristics similar to normal articular cartilage. Long-term investigations are needed to determine the durability of the repair produced with this technique.     

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.