Cell-free repair of small cartilage defects in the Goettinger minipig: which defect size is possible?

PURPOSE: Cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with cell-free collagen type-I gel plugs of three different sizes. METHODS: In 6 adult Goettinger minipigs, three full-thickness chondral defects were created in the trochlear groove of one knee of the hind leg. These defects were treated with a cell-free collagen type-I gel plug of 8, 10, or 12 mm diameter. All animals were allowed unlimited weight bearing. After 1 year, the animals were killed. Immediately after recovery, a non-destructive biomechanical testing was performed. The repair tissue quality was evaluated immunohistologically, collagen type-II protein was quantified, and a semiquantitative score (O'Driscoll score) was calculated. RESULTS: After 1 year, a high number of cells migrated into the initially cell-free collagen gel plugs and a hyaline-like repair tissue had been created. The O'Driscoll scores were: 8 mm, 21.2 (SD, 2.8); 10 mm, 21.5 (SD, 1.6); and 12 mm, 22.3 (SD, 1.0). The determination of the e-modulus, creep and relaxation revealed that mechanical properties of the two smaller defects were closer to unaffected hyaline cartilage. CONCLUSIONS: As cell-free collagen type-I gel plugs of all three different sizes created hyaline-like repair tissue, this system seems suitable for the treatment of even larger defects.

Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin.

Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti-H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.

Expression of ion channels of the TRP family in articular chondrocytes from osteoarthritic patients: changes between native and in vitro propagated chondrocytes.

The maintenance of a differentiated chondrocyte phenotype is influenced by several factors of which signal transduction of extracellular stimuli through the cell membrane is of major interest. One important group of membrane-bound proteins which are involved in transmembrane signal transduction are ion channels. Human articular chondrocytes were obtained from osteoarthritic femoral condyles. Cells were released from the surrounding matrix and cultivated under standard conditions. We investigated gene expression of 12 members of the TRP ion channel family of freshly prepared (passage 0; P0) and in vitro propagated human articular chondrocytes (passage 2; P2) using conventional and real-time PCR (RT-PCR). In addition, the protein appearance of four TRP channels was demonstrated by immunofluorescence and western blotting. Chondrocyte differentiation was monitored by quantification of collagen type-II, type-I, and aggrecan gene expression. By conventional PCR, 8 channels could be detected, of which some displayed a heterogeneous PCR pattern. RT-PCR quantification revealed that TRPC1 was expressed on the same level in P0 and P2 chondrocytes while gene expression of TRPC3 and TRPC6 was elevated in passage 2 cells. TRPM5, TRPM7, and TRPV1 displayed an enhanced gene expression in freshly isolated chondrocytes. Immunofluorescence signal intensity of all four investigated TRP proteins was consistent with the corresponding gene expression data. In the present study, a correlation between the appearance of some members of the TRP ion channel family and the state of de-differentiation of osteoarthritic articular chondrocytes was shown. A possible direct involvement in the process of chondrocyte de-differentiation has to be investigated in further studies.

A novel rat tail collagen type-I gel for the cultivation of human articular chondrocytes in low cell density.

Collagen type-I matrix systems have gained growing importance as a cartilage repair device. However, most of the established matrix systems use collagen type-I of bovine origin seeded in high cell densities. Here we present a novel collagen type-I gel system made of rat tail collagen for the cultivation of human chondrocytes in low cell densities. Rat tail collagen type-I gel (CaReS, Arthro Kinetics, Esslingen, Germany) was seeded with human passage 2 chondrocytes in different cell densities to evaluate the optimal cell number. In vitro, the proliferation factor of low density cultures was more than threefold higher compared with high density cultures. After 6 weeks of in vitro cultivation, freshly prepared chondrocytes with an initial cell density of 2x10(5) cells/mL showed a proliferation factor of 33. A cell density of 2x10(5) cells/mL was chosen for in vitro and in vivo cultivation using the common nude mouse model as an in vivo system. Chondrocytes stayed viable as a Live/Dead fluorescence assay and TUNEL staining revealed. During in vitro cultivation, passage 0 cells partly dedifferentiated morphologically. In vivo, passage 0 cells maintained the chondrocyte phenotype and demonstrated an increased synthesis of collagen type-II protein and gene expression compared to passage 2 cells. Passage 2 cells did not redifferentiate in vivo. Cultivating a cell-seeded collagen gel of bovine origin as a control (AtelocollagenTM, Koken, Tokyo, Japan) did not lead to superior results with regard to cell morphology, col-II protein production and col-II gene expression. With the CaReS collagen gel system the best quality of repair tissue was obtained by seeding freshly isolated chondrocytes.

In vivo cultivation of human articular chondrocytes in a nude mouse-based contained defect organ culture model.

The nude mouse model is an established method to cultivate and investigate tissue engineered cartilage analogues under in vivo conditions. One limitation of this common approach is the lack of appropriate surrounding articular tissues. Thus the bonding capacity of cartilage repair tissue cannot be evaluated. Widely applied surgical techniques in cartilage repair such as conventional and three-dimensional autologous chondrocyte implantation (ACI) based on a collagen gel matrix cannot be included into nude mouse studies, since their application require a contained defect. The aim of this study is to apply an organ culture defect model for the in vivo cultivation of different cell-matrix-constructs. Cartilage defects were created on osteochondral specimens which had been harvested from 10 human knee joints during total knee replacement. Autologous chondrocytes were isolated from the cartilage samples and cultivated in monolayer until passage 2. On each osteochondral block defects were treated either by conventional ACI or a collagen gel seeded with autologous chondrocytes, including a defect left empty as a control. The samples were implanted into the subcutaneous pouches of nude mice and cultivated for six weeks. After retrieval, the specimens were examined histologically, immunohistochemically and by cell morphology quantification. In both, ACI and collagen gel based defect treatment, a repair tissue was formed, which filled the defect and bonded to the adjacent tissues. The repair tissue was immature with low production of collagen type II. In both groups redifferentiation of chondrocytes remained incomplete. Different appearances of interface zones between the repair tissue and the adjacent cartilage were found. The presented contained defect organ culture model offers the possibility to directly compare different types of clinically applied biologic cartilage repair techniques using human articular tissues in a nude mouse model.

Electrical potentials derived from articular cartilage: the significance of polarization potentials.

Changes of electrical potentials in cartilage tissue under mechanical compression have been detected in several studies. As polarization potentials are known to occur at the interface between metals and electrolytes, the question remains open whether the measured electrical potentials in relation to mechanical compression are interfered with polarization potentials. Using a porcine model, whole knee joints were explanted and exposed to uniaxial loading of up to 250 N. Under similar conditions, a tube filled with normal saline was prepared with three gold-plated electrodes. Changes of voltage derived from the electrodes placed in normal saline could be detected only when the force was applied instantly by a hydraulically controlled pump. In comparison, mechanically induced electrical potentials could be derived from cartilage tissue when exposed to both sudden force and force induced more slowly by an electric engine. While the electrical response derived from cartilage tissue correlated with the extent of the applied force, there was no such correlation between the potential changes from normal saline and the applied mechanical force. In conclusion, polarization potentials derived from metal electrodes in contact with electrolyte solution are pressure dependent. However, those electrical potential changes obtained from the cartilage tissue under compressive force revealed no obvious influence by polarization potentials.

Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest.

During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.

Histone gene expression and chromatin structure during spermatogenesis.

The chromatin of male germ cells is restructured throughout spermatogenesis. Analysis of differential histone protein patterns at specific stages of spermatogenesis may contribute towards an understanding of the changes in chromatin structure and function during this differentiation process. The most striking changes in histone patterns occur at the stage of pachytene spermatocytes when most of the linker H1 histones are replaced by the testis specific subtype H1t. In addition, replacement of core histone subtypes is observed at this stage. These structural changes precede the reorganization of chromatin at haploid stages when histones are replaced first by transition proteins and then by protamines.

[Electromagnetic fields, electric current and bone healing - what is the evidence?]. / Elektromagnetische Felder, elektrischer Strom und Knochenheilung: was ist gesichert?

For more than 30 years the potential effects of electrical stimulation on bone healing have been investigated. Up to now this therapy is controversial and not established as a standardised treatment option. This systematic review and metaanalysis focuses on the potential effects of electromagnetic fields and high-frequency electric fields on bony healing. In a systematic literature search randomised clinical trials were identified and analysed. Those studies with the primary endpoint "rate of bony healing" were combined in a metaanalysis which was performed with the "random effects" model. We found a total of 14 randomised clinical trials. These studies included a total of 915 patients. The majority of these studies used pulsed electromagnetic fields (PEMF). Out of the 14 studies nine were suitable for the metaanalysis which revealed a cumulative odds ratio of 3.5 and a 95 % confidence interval of 1.94-6.3. When performing a subgroup analysis a statistically significant result could not be confirmed by the studies with a higher methodological quality. In view of the heterogeneous physical parameters with different frequencies, time course, flux densities and in view of the methodological deficits, a general conclusion seems difficult. Recommendations or standards of therapy are so far not available.

[Tissue engineering of cartilage replacement material - mechanical stimulation in the in-vitro cultivation of human chondrocytes]. / Tissue Engineering von Knorpelersatzgewebe - Mechanische Stimulation in der In-vitro-Kultivierung von humanen Chondrozyten.

The treatment of cartilage defects remains a major problem in orthopaedics. With regard to cartilage tissue engineering, the reimplantation of pre-cultivated chondrocytes in the form of a chondrocyte graft is a promising alternative to conventional methods. Clinical practice requires this MACT procedure (matrix-associated autologous chondrocyte transplantation) to produce a biocompatible replacement tissue with adequate mechanical properties. Mechanical stimulation has the capacity to improve the quality of these cell-seeded biomaterials. By altering chondrocytes' cellular activities, the biological and biomechanical properties of cartilage replacement tissue can be modulated. Different systems are used for this purpose, e.g. shear, perfusion, hydrostatic pressure or compression. The mechanisms, biological effects, chances and problems of the techniques are presented and assessed. Among the stimulating techniques considered are systems that apply indirect and direct shear forces such as spinner flasks, rotating-wall bioreactors, direct tissue shear and perfusion culture systems. The application of hydrostatic pressure or compression may be brought about by either static or dynamic loading systems. Compressive loading is considered in the light of both its short- and long-term effects; additionally two exemplified systems are discussed in detail. However, despite promising approaches and seemingly favourable tissue characteristics, the in vitro culturing of functional cartilage replacement tissue with cartilage-like mechanical and biological characteristics still remains elusive. Furthermore, controlling, monitoring and regulating culturing conditions are general biotechnological requirements of a standardised in vitro cultivation. Among these, different aspects such as aseptic operation, media supplementation, nutrient and gas exchange, temperature and humidity control are considered.

[Optical coherence tomography (OCT) to evaluate cartilage tissue engineering]. / Die optische Kohärenztomografie (OCT) zur Darstellung der Regenerateigenschaften im Tissue Engineering von Knorpelgewebe.

AIM: The aim of this study was to investigate hyaline cartilage defects treated with cell-seeded artificial matrix systems (two different collagen type I gels) with the method of optical coherence tomography (OCT) and to correlate the results with conventional histological and immunocytochemical staining. METHOD: Osteochondral blocks were harvested from 20 patients undergoing total knee replacement and trimmed to 2 x 2 cm. Under sterile conditions, chondral defects of 8 mm diameter were either filled with a collagen type I gel plug seeded with autologous chondrocytes (2 x 10 (5)/mL gel), or with a corresponding gel plug which was stabilised by a 20-fold compression. Of each group, 5 specimens were cultivated for 6 weeks under standardised in vitro conditions (37 degrees C, 5 % CO (2), humidified atmosphere), while the remaining 5 specimens were implanted subcutaneously in nude mice (BALBc -/-). Immediately after recovery, the repair tissue and bonding zones were investigated by OCT. Subsequently, specimens were decalcified and investigated by H&E staining and collagen type II immunostaining. The results of OCT and conventional staining were correlated. RESULTS: By OCT, repair tissue could be investigated up to 1.6 mm in depth, physically limited by the utilised OCT system. In the denser hyaline cartilage regions, OCT resolution was reduced. Regardless of cultivation (in vitro or nude mouse), ultrastructural features of the repair tissue could be demonstrated. In particular, ultrastructural differences between the two investigated collagen gels could be detected. Moreover, the bonding region between repair tissue and hyaline cartilage could be evaluated by OCT investigation. The results of the OCT measurements were confirmed by H&E and collagen type II immunostaining. CONCLUSION: By OCT, repair tissue generated by the treatment of cartilage defects with tissue-engineered matrix systems could be evaluated in vitro. Future studies may show if repair tissue quality may be monitored in situ by OCT.

[Treatment of patellofemoral cartilage defects utilizing a 3D collagen gel: two-year clinical results]. / Die Behandlung femoropatellarer Knorpelschäden mit einem dreidimensionalen Kollagengel: Klinische Ergebnisse im Zwei-Jahres-Verlauf.

AIM: Here we present first clinical results of the treatment of patellofemoral cartilage lesions with a 3D collagen gel (CaReS). METHODS: Isolated patellofemoral cartilage lesions in 14 patients (13 patellar, 1 trochlear) were treated by matrix-based ACT using a collagen gel seeded with autologous chondrocytes. In this study, only those patients were taken into account who complied with the inclusion criteria of the German working group "tissue regeneration and tissue substitutes". Data were analysed in accordance with the ICRS criteria and the Brittberg score. We present the 2-year postoperative clinical follow-up. RESULTS: After 2 years the ICRS-IKDC and Brittberg scores revealed a statistically significant improvement. The preoperative rating of the objectiveIKDC and functional ICRS score improved from 0/14 and 2/14 (14.3%) to 11/14 (78.6%) in the categories A/B and I/II, respectively. 11 Patients(78.6%) judged the clinical outcome as excellent or good. The subjective IKDC score improved from 32.4 +/- 8.4 prior to operation to 67.8 +/- 27.4 at the 2-years follow-up. CONCLUSION: The use of 3D matrix systems could represent a promising improvement of conventional ACT in the treatment of patellofemoral cartilage lesions, but the results have to be verified by long-term investigations.

[First clinical experiences with a novel 3D-collagen gel (CaReS) for the treatment of focal cartilage defects in the knee]. / Erste klinische Erfahrungen mit einem neuartigen dreidimensionalen Kollagengel (CaReS) zur Behandlung fokaler Knorpeldefekte am Kniegelenk.

AIM: In this prospective study we present a novel technique of matrix-based chondrocyte transplantation for the treatment of focal cartilage defects. METHOD: 22 patients with chondral or osteochondral femoral defects were treated with 3D-collagen type-I gel seeded with autologous chondrocytes (CaReS). Overall handling of this novel method is described, and results after 2 years follow-up with regard to the ICRS-score are presented. RESULTS: 14 patients were treated because of a focal chondral lesion, in 8 cases subchondral bone had to be reestablished. The average defect size was 6 cm(2). In all cases cell-seeded matrix samples could be fixed with fibrin glue without any problems. The average surgery time was 69 min, length of the surgical approach was 8.2 cm. No surgery-specific problems had to be observed. A significant improvement in IKDC-score, functional score and overall rating after 3 or 6 months up to 2 years follow-up was detected. After 2 years post-OP 13 Patients were evaluated and matrix-based defect treatment by CaReS was rated good to excellent by 84.6 % of the patients. CONCLUSION: Defect treatment with cell-seeded 3D-collagen gel is easy to handle with a good clinical outcome after 2 years follow-up. Further investigations with higher patient number, longer follow-up time and histological and biomechanical sample analysis are needed in order to establish this method as an improvement of conventional ACT.

[Biomechanical tests of a new scaffold for the cultivation of chondrocytes]. / Biomechanische Testung einer neuen Trägermatrix für die Kultivierung von Chondrozyten.

AIM: Scaffolds for the cultivation of chondrocytes are of increasing importance. So far, only little is known about their biomechanical properties. The present preliminary study addresses the biomechanical characteristics of a new collagen type I scaffold for the cultivation of chondrocytes. MATERIAL AND METHODS: Human chondrocytes were amplified in a monolayer and then cultivated in a 3D-scaffold over a period of up to 6 weeks. The biomechanical tests addressed the properties under uniaxial compression including stiffness and viscoelastic characteristics (creep and retardation). The obtained values were normalized against the thickness of the specimens and expressed as ratios. In addition, we present histological and quantitative PCR results (for collagen type II and aggrecan). RESULTS: The maximum force (or penetration force) revealed its highest values after a period of seven days. At this time the median value was 40 mN/mm. In the following period, a marked drop of the values was observed (19.8 mN/mm). With respect to the creep properties, we did not find any major changes over the period of six weeks. The median values were between 0.24 and 0.29 mm/mm. There were no significant differences between the samples seeded with chondrocytes and those which served as controls. A re-expansion of the samples was found with median values between 0.026 and 0.049 mm/mm (retardation). However, the original thickness was not reached after a period of 30 seconds with relief of the strain. Again, major differences of the values with respect to the duration of cultivation were not observed. Light microscopy revealed collagen type II and proteoglycans only in the pericellular region. CONCLUSION: In this study not all of the biomechanical properties of the cultivated tissue were investigated. The limitation of the tests to stiffness and viscoelastic properties was reasonable in view of a potential routine use. In addition, it may facilitate a comparison between different matrix systems. In our study, the cultivation of cells within the collagen matrix did not alter the mechanical properties of the scaffold.

Expression of mRNA and protein of nucleoproteins during human spermiogenesis.

The most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, which are the basic nuclear proteins of mature spermatozoa. A first step in this exchange is the displacement of histones by transition proteins (TP). Our study demonstrates, for the first time, the sequential expression of the testis-specific histone (H1t) and the transition proteins (TP1 and TP2) during normal human spermatogenesis. H1t mRNA could only be detected in the cytoplasm of mid and late pachytene spermatocytes. Concomitant with the onset of H1t transcription, the H1t protein appeared in the nuclei of pachytene spermatocytes and remain as a nuclear protein constituent up to step 5 spermatids. While transition protein 1 gene TNP-1 mRNA was present in spermatids from step 2 to early step 4, the TP1 protein occured, with temporal delay, in the nuclei of step 3 and step 4 spermatids. The TP2 protein was observed in the nuclei of spermatids from step 1 to step 5. The transition protein 2 gene TNP-2 mRNA was only detected by reverse transcription-polymerase chain reaction, but not on paraffin sections. These data demonstrate a strong temporal association between H1t gene transcription and synthesis of the H1t protein. Since the TP1 protein appeared with temporal delay we can assume that the corresponding TNP-1 mRNA is translationally delayed.

Histones: genetic diversity and tissue-specific gene expression.

Histones are the major protein constituents of the chromatin of eukaryotic cell nuclei. This group of basic proteins is extremely conserved throughout evolution and includes five classes termed H1, H2A, H2B, H3 and H4. In mammals, each of these classes except H4 is subdivided into several subtypes. The most divergent class of histones is the H1 protein family, which consists of seven different subtypes, termed H1.1-H1.5, H1 degree, and H1t. The subtypes H1.2 and H1.4 are found in most somatic cell nuclei, whereas H1 degree is found in several differentiated tissues, and H1t is restricted to mammalian testicular cells. Similarly, core histone subtypes replacing the major forms of H2A, H2B or H3 have been described. Biochemical analysis of protein and RNA from different tissues and cell lines demonstrates varied patterns of expression of individual histone subtype genes. Moreover, antibodies against specific histone subtypes and in situ hybridization with subtype-specific probes indicate that the expression of histone subtype genes is in several cases modulated in a tissue-specific manner. This is particularly evident at the different stages of spermatogenesis when chromatin undergoes substantial reorganization, which finally results in the highly condensed state of chromatin of the mature sperm head.