Tomasz Trzeciak

The role of growth factors in stem cell-directed chondrogenesis: a real hope for damaged cartilage regeneration

PurposeThe use of stem cells in regenerative medicine offers hope to treat numerous orthopaedic disorders, including articular cartilage defects. Although much research has been carried out on chondrogenesis, this complicated process is still not well understood and much more research is needed. The present review provides an overview of the stages of chondrogenesis and describes the effects of various growth factors, which act during the multiple steps involved in stem cell-directed differentiation towards chondrocytes.MethodsThe current literature on stem cell-directed chondrogenesis, in particular the role of members of the transforming growth factor-β (TGF-β) superfamily—TGF-βs, bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs)—is reviewed and discussed.ResultsNumerous studies have reported the chondrogenic potential of both adult- and embryonic-like stem cells and the role of growth factors in programming differentiation of these cells towards chondrocytes. Mesenchymal stem cells (MSCs) are adult multipotent stem cells, whereas induced pluripotent stem cells (iPSC) are reprogrammed pluripotent cells. Although better understanding of the processes involved in the development of cartilage tissues is necessary, both cell types may be of value in the clinical treatment of cartilage injuries or osteoarthritic cartilage lesions.ConclusionsMSCs and iPSCs both present unique characteristics. However, at present, it is still unclear which cell type is most suitable in the treatment of cartilage injuries.

The role of adipocytokines in the pathogenesis of knee joint osteoarthritis.

Osteoarthritis (OA) is one of the most common causes of musculoskeletal disability in the world. Traditionally, it has been thought that obesity contributes to the development and progression of OA by increased mechanical load of the joint structures. Nevertheless, studies have shown that adipose tissue-derived cytokines (adipocytokines) are a possible link between obesity and OA. Furthermore, according to recent findings, not only articular cartilage may be the main target of these cytokines but also the synovial membrane, subchondral bone and infrapatellar fat pad may be encompassed in the process of degradation. This review presents the most recent reports on the contribution of adipocytokines to the knee joint cartilage degradation, osteophyte formation, infrapatellar fat pad alterations and synovitis.

Osteoarthritis and telomere shortening

Osteoarthritis is the most common disease of joints caused by degradation of articular cartilage and subchondral bone. It is classified as primary form with unknown cause and as secondary form with known etiology. Genetic and epigenetic factors interact with environmental factors and contribute to the development of primary osteoarthritis. Thus far, many polymorphisms associated with osteoarthritis have been identified and recent studies also indicate the involvement of epigenetic factors (e.g., telomere shortening) in the initiation of this disorder. Accelerated shortening of telomeres was detected in osteoarthritis and other age-related diseases. Studies revealed that telomere length is severely reduced in blood leukocytes and chondrocytes of patients with osteoarthritis, and this may contribute to the initiation and development of osteoarthritis, whose major cause is still unknown.

Directed differentiation of induced pluripotent stem cells into chondrogenic lineages for articular cartilage treatment

In recent years, increases in the number of articular cartilage injuries caused by environmental factors or pathological conditions have led to a notable rise in the incidence of premature osteoarthritis. Osteoarthritis, considered a disease of civilization, is the leading cause of disability. At present, standard methods for treating damaged articular cartilage, including autologous chondrocyte implantation or microfracture, are short-term solutions with important side effects. Emerging treatments include the use of induced pluripotent stem cells, a technique that could provide a new tool for treatment of joint damage. However, research in this area is still early, and no optimal protocol for transforming induced pluripotent stem cells into chondrocytes has yet been established. Developments in our understanding of cartilage developmental biology, together with the use of modern technologies in the field of tissue engineering, provide an opportunity to create a complete functional model of articular cartilage.

MicroRNAs: Important Epigenetic Regulators in Osteoarthritis

Multiple mechanisms are implicated in the development of primary osteoarthritis (OA), in which genetic and epigenetic factors appear to interact with environmental factors and age to initiate the disease and stimulate its progression. Changes in expression of microRNAs (miRs) contribute to development of osteoarthritis. Numerous miRs are involved in cartilage development, homeostasis and degradation through targeting genes expressed in this tissue. An important regulator of gene expression in human cartilage is miR-140, which directly targets a gene coding aggrecanase ADAMTS-5, that cleaves aggrecan in cartilage. This miR is considered a biological marker for cartilage and its level significantly decreases in OA cartilage. On the other hand, increased expression of miR-146a in early OA inhibits two other cartilage-degrading enzymes: MMP13 and ADAMTS4, and may provide a useful tool in developing treatments for OA. The COL2A1 gene, encoding collagen type II, which is the most abundant structural protein of the cartilage, is silenced by miR-34a and activated by miR-675. Every year, new targets of cartilage miRs are validated experimentally and this opens new possibilities for new therapies that control joint destruction and stimulate cartilage repair. At the same time development of next-generation sequencing technologies allows to identify new miRs involved in cartilage biology.

Bilateral radial agenesis with absent thumbs, complex heart defect, short stature, and facial dysmorphism in a patient with pure distal microduplication of 5q35.2-5q35.3

BackgroundA partial duplication of the distal long arm of chromosome 5 (5q35-- > qter) is known to be associated with a distinct phenotype referred to as Hunter-McAlpine syndrome. Clinical spectrum of this disorder mainly consists of mental retardation, microcephaly, short stature, skeletal anomalies, and craniofacial dysmorphism featuring flat facies, micrognathia, large, low-set dysplastic ears, hypertelorism, almond-shaped, down-slanted palpebral fissures, epicanthal folds, small nose, long philtrum, small mouth, and thin upper lip. Less frequent remarkable findings include craniosynostosis, heart defect, hypoplastic phalanges, preaxial polydactyly, hypospadias, cryptorchidism, and inguinal hernia. In most patients with a partial duplication of 5q the aberration occurred due to an inherited unbalanced translocation, therefore the phenotype was not reflective of pure trisomy 5q.Case presentationWe report on a 9.5-year-old boy with some feature of Hunter-McAlpine syndrome including short stature, complex heart defect (dextrocardia, dextroversion, PFO), bilateral cryptorchidism, hypothyroidism, and craniofacial dysmorphism. Additionally, bilateral radial agenesis with complete absence of Ist digital rays, ulnar hypoplasia with bowing, choroidal and retinal coloboma, abnormal biliary vesicle were identified, which have never been noted in 5q trisomy patients. Karyotype analysis, sequencing and MLPA for TBX5 and SALL4 genes were unremarkable. Array comparative genomic hybridization detected a duplication on 5q35.2-5q35.3, resulting from a de novo chromosomal rearrangement. Our proband carried the smallest of all previously reported pure distal 5q trisomies encompassing terminal 5.4-5.6 Mb and presented with the most severe limb malformation attributed to the increased number of distal 5q copies.ConclusionsWe postulate that a terminal distal trisomy of 5q35.2-5q35.3, which maps 1.1 Mb telomeric to the MSX2 gene is causative for both radial agenesis and complex heart defect in our proband. A potential candidate gene causative for limb malformation in our proband could be FGFR4, which maps relatively in the closest position to the chromosomal breakage site (about 1.3 Mb) from all known 5q duplications. Since the limb malformation as well as the underlying genetic defect are distinct from other 5q trisomy patient we propose that a position effect resulting in altered long-range regulation of the FGFR4 (alternatively MSX2) may be responsible for the limb malformation in our proband.

Bioimaging: An Useful Tool to Monitor Differentiation of Human Embryonic Stem Cells into Chondrocytes

To improve the recovery of damaged cartilage tissue, pluripotent stem cell-based therapies are being intensively explored. A number of techniques exist that enable monitoring of stem cell differentiation, including immunofluorescence staining. This simple and fast method enables changes to be observed during the differentiation process. Here, two protocols for the differentiation of human embryonic stem cells into chondrocytes were used (monolayer cell culture and embryoid body formation). Cells were labeled for markers expressed during the differentiation process at different time points (pluripotent: NANOG, SOX2, OCT3/4, E-cadherin; prochondrogenic: SOX6, SOX9, Collagen type II; extracellular matrix components: chondroitin sulfate, heparan sulfate; beta-catenin, CXCR4, and Brachyury). Comparison of the signal intensity of differentiated cells to control cell populations (articular cartilage chondrocytes and human embryonic stem cells) showed decreased signal intensities of pluripotent markers, E-cadherin and beta-catenin. Increased signal intensities of prochondrogenic markers and extracellular matrix components were observed. The changes during chondrogenic differentiation monitored by evaluation of pluripotent and chondrogenic markers signal intensity were described. The changes were similar to several studies over chondrogenesis. These results were confirmed by semi-quantitative analysis of IF signals. In this research we indicate a bioimaging as a useful tool to monitor and semi-quantify the IF pictures during the differentiation of hES into chondrocyte-like.

Induced Pluripotent and Mesenchymal Stem Cells as a Promising Tool for Articular Cartilage Regeneration

The application of stem cells in regenerative medicine has recently become a rapidly growing field, holding promise for combating a number of orthopedic disorders including osteodegenerative ones (osteoporosis and osteoarthritis). Although the differentiation of stem cells into chondrocytes is now intensively investigated on a laboratory scale, implementing the laboratory protocols in clinical practice requires a scale-up culture. In order to apply this technique many aspects of stem cell bioprocessing such as optimal culture conditions for anchoragedependent or anchorage-independent cells and the type of culture must be taken into account. The presence of microcarriers and/or scaffolds for adherent cells is essential, since they provide a three-dimensional microenvironment indispensable for cell growth. For treatment of osteoarthritis, induced pluripotent stem cells and mesenchymal stem cells seem to be the best choice. Although, the scale-up culture using stem cells has been intensively investigated on a laboratory scale, the scale-up culture for clinical application still requires further technical improvements.In this review stem cell bioprocessing including the use of biomaterials, bioreactors, and factors affecting this process, as well as scale-up culture of induced Pluripotent and mesenchymal stem cells were presented and discussed.

Variable expressivity of the phenotype in two families with brachydactyly type E, craniofacial dysmorphism, short stature and delayed bone age caused by novel heterozygous mutations in the PTHLH gene

Brachydactyly refers to shortening of digits due to hypoplasia or aplasia of bones forming the hands and/or feet. Isolated brachydactyly type E (BDE), which is characterized by shortened metacarpals and/or metatarsals, results in a small proportion of patients from HOXD13 or PTHLH mutations, although in the majority of cases molecular lesion remains unknown. BDE, like other brachydactylies, shows clinical heterogeneity with highly variable intrafamilial and interindividual expressivity. In this study, we investigated two Polish cases (one familial and one sporadic) presenting with BDE and additional symptoms due to novel PTHLH mutations. Apart from BDE, the affected family showed short stature, mild craniofacial dysmorphism and delayed bone age. Sanger sequencing of PTHLH revealed a novel heterozygous frameshift mutation c.258delC(p.N87Tfs*18) in two affected individuals and one relative manifesting mild brachydactyly. The sporadic patient, in addition to BDE, presented with craniofacial dysmorphism, normal stature and bone age, and was demonstrated to carry a de novo heterozygous c.166C>T(p.R56*) mutation. Our paper reports on the two novel truncating PTHLH variants, resulting in variable combination of BDE and other symptoms. Data shown here expand the knowledge on the phenotypic presentation of PTHLH mutations, highlighting significant clinical variability and incomplete penetrance of the PTHLH-related symptoms.

New intermediate phenotype between MED and DD caused by compound heterozygous mutations in the DTDST gene.

DTDST mutations cause a spectrum of diastrophic dysplasia disorders characterized by defects of proteoglycans sulfation. Reduction of sulfate/chloride antiporter activity is manifested by lower sulfate uptake and depends on a combination of mutations in DTDST. We analyzed a family with an autosomal recessive form of bone dysplasia. Three affected brothers from this family are compound heterozygotes for C653S/A715V mutations. We classified their phenotype as a new intermediate form between diastrophic dysplasia and multiple epiphyseal dysplasia, manifested by shortening of stature, metatarsus adductus/club foot, mild brachydactyly, proximally placed thumbs and clinodactyly of the fifth fingers. Radiographs document platyspondyly most marked in the lower thoracic and upper lumbar spine, epiphyseal dysplasia affecting predominantly the femoral heads, widening of the metaphyses, narrow growth cartilage and multilayered patellae. Exaggerated lesser trochanters of femur, that is, “monkey wrench” sign, elevated greater trochanters, thin upper pubic rami, grossly normal carpal/tarsal bones and severe, early onset osteoarthritis were other notable features.