A composite hydrogel-3D printed thermoplast osteochondral anchor as example for a zonal approach to cartilage repair: in vivo performance in a long-term equine model.

Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into the chondral part of the construct over which two layers of a thiol-ene cross-linkable hyaluronic acid/poly(glycidol) hybrid hydrogel (HA-SH/P(AGE-co-G)) were fabricated. The top layer contained Articular Cartilage Progenitor Cells (ACPCs) derived from the superficial layer of native cartilage tissue, the bottom layer contained mesenchymal stromal cells (MSCs). The chondral part of control constructs were homogeneously filled with MSCs. After six months in vivo, microtomography revealed significant bone growth into the anchor. Histologically, there was only limited production of cartilage-like tissue (despite persistency of hydrogel) both in zonal and non-zonal constructs. There were no differences in histological scoring; however, the repair tissue was significantly stiffer in defects repaired with zonal constructs. The sub-optimal quality of the repair tissue may be related to several factors, including early loss of implanted cells, or inappropriate degradation rate of the hydrogel. Nonetheless, this approach may be promising and research into further tailoring of biomaterials and of construct characteristics seems warranted.

Reduced hypertrophy in vitro after chondrogenic differentiation of adult human mesenchymal stem cells following adenoviral SOX9 gene delivery.

BACKGROUND: Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral gene-delivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro. METHODS: First generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 × 102 infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. RESULTS: SOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels. CONCLUSION: Adenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.

A definition of bioinks and their distinction from biomaterial inks.

Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

Silver and copper addition enhances the antimicrobial activity of calcium hydroxide coatings on titanium.

Electrochemically assisted deposition of Ca(OH)2 (Portlandite) coatings on titanium surfaces has been proven as a promising method to provide the substrate with a most desirable combination of significant bacterial growth reduction on one hand and good biocompatibility on the other. Due to the rapid in vivo transformation of Ca(OH)2 to hydroxyapatite, the antimicrobial activity will be an ephemeral property of the coating when implanted into the human body. In this study, the ability to reduce bacterial growth of such portlandite coatings was significantly enhanced by an ionic modification with copper and silver ions. Antibacterial tests revealed a noticeably elevated reduction of bacterial growth, especially for copper and even at a relatively low copper content of about 0.3 wt.%. In addition, the cytocompatibility, a crucial prerequisite for potential in vivo biocompatibility, of the copper-modified coating was comparable to pure calcium hydroxide coatings.

The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model.

OBJECTIVE: To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. METHODS: After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaP scaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. RESULTS: The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the +P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P < 0.0001). CONCLUSIONS: The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the -P group integrated well with the recipient bone. The study stresses the importance of long-term in vivo studies to assess the efficacy of cartilage repair techniques.

Electrochemically assisted deposition of strontium modified magnesium phosphate on titanium surfaces.

Electrochemically assisted deposition was utilized to produce ceramic coatings on the basis of magnesium ammonium phosphate (struvite) on corundum-blasted titanium surfaces. By the addition of defined concentrations of strontium nitrate to the coating electrolyte Sr(2+) ions were successfully incorporated into the struvite matrix. By variation of deposition parameters it was possible to fabricate coatings with different kinetics of Sr(2+) into physiological media, whereas the release of therapeutically relevant strontium doses could be sustained over several weeks. Morphological and crystallographic examinations of the immersed coatings revealed that the degradation of struvite and the release of Sr(2+) ions were accompanied by a transformation of the coating to a calcium phosphate based phase similar to low-crystalline hydroxyapatite. These findings showed that strontium doped struvite coatings may provide a promising degradable coating system for the local application of strontium or other biologically active metal ions in the implant-bone interface.

Real-time measurement of protein adsorption on electrophoretically deposited hydroxyapatite coatings and magnetron sputtered metallic films using the surface acoustic wave technique.

Surface acoustic wave (SAW) biosensors are highly sensitive for mass binding and are therefore used to detect protein-protein and protein-antibody interactions. Whilst the standard surface of the chips is a thin gold film, measurements on implant- or bone-like surfaces could significantly enhance the range of possible applications for this technique. The aim of this study was to establish methods to coat biosensor chips with Ti, TiN, and silver-doped TiN using physical vapor deposition as well as with hydroxyapatite by electrophoresis. To demonstrate that protein adsorption can be detected on these surfaces, binding experiments with fibronectin and fibronectin-specific antibodies have been performed with the coatings, which successfully proved the applicability of PVD and EPD for SAW biosensor functionalization.

Current treatment limitations in age-related macular degeneration and future approaches based on cell therapy and tissue engineering.

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruch's membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein.

Dual setting α-tricalcium phosphate cements.

An extension of the application of calcium phosphate cements (CPC) to load-bearing defects, e.g. in vertebroplasty, would require less brittle cements with an increased fracture toughness. Here we report the modification of CPC made of alpha-tricalcium phosphate (α-TCP) with 2-hydroxyethylmethacrylate (HEMA), which is polymerised during setting to obtain a mechanically stable polymer-ceramic composite with interpenetrating organic and inorganic networks. The cement liquid was modified by the addition of 30-70 % HEMA and ammoniumpersulfate/tetramethylethylendiamine as initiator. Modification of α-TCP cement paste with HEMA decreased the setting time from 14 min to 3-8 min depending on the initiator concentration. The 4-point bending strength was increased from 9 MPa to more than 14 MPa when using 50 % HEMA, while the bending modulus decreased from 18 GPa to approx. 4 GPa. The addition of ≥50 % HEMA reduced the brittle fracture behaviour of the cements and resulted in an increase of the work of fracture by more than an order of magnitude. X-ray diffraction analyses revealed that the degree of transformation of α-TCP to calcium deficient hydroxyapatite was lower for polymer modified cements (82 % for polymer free cement and 55 % for 70 % HEMA) after 24 h setting, while the polymerisation of HEMA in the cement liquid was quantitative according to FT-IR spectroscopy. This work demonstrated the feasibility of producing fracture resistant dual-setting calcium phosphate cements by adding water soluble polymerisable monomers to the liquid cement phase, which may be suitable for an application in load-bearing bone defects.

P2RX7 genotype association in severe sepsis identified by a novel Multi-Individual Array for rapid screening and replication of risk SNPs.

BACKGROUND: Functional single nucleotide polymorphisms (SNPs) are relevant to individual therapeutic approaches and may play a role in disease susceptibility. Genome-wide scans, which are now widely applied to detect disease-associated SNPs, provide only limited evidence about SNP associations. Their usefulness as disease markers requires appropriate phenotype analysis and retesting of the gene providing SNP information. Larger data sets of thousands of samples are necessary to confirm the suggested SNPs. METHODS: We applied a newly established microarray-based technology that significantly accelerates and simplifies such studies. A tailor-made microarray surface chemistry, sample/probe immobilization and a primer extension reaction are central to the multi-individual array (MIA) platform, which simultaneously identifies the same variable nucleotide in thousands of samples. The set of SNPs to be typed for is highly flexible and can be adapted to the demands of defined clinical questions. RESULTS: A MIA-SNP analysis of functional SNPs in the P2RX7 calcium channel is presented. One risk genotype has been verified by functional analysis using patch clamping. Two clinically relevant genotypes composed of 5 functional SNPs in the P2RX7 gene have been identified in patients with severe sepsis and septic shock, whereas no significant association has been found in patient suffering from hemophagocytic syndromes. CONCLUSIONS: These results support a functional SNP genotyping of the P2RX7 gene in patients at risk of severe sepsis following surgical trauma.

Comparison of coatings from reactive star shaped PEG-stat-PPG prepolymers and grafted linear PEG for biological and medical applications.

Grafting of poly(ethylene glycol) (PEG) is a common strategy for reducing nonspecific interactions of surfaces with proteins. We have used grafting at "cloud point" solution conditions that ensures maximum grafting density of linear methoxy terminated PEG-aldehyde (mPEG-ald, M(w) = 5000 and 30000). In an alternative approach, surfaces were modified with layers prepared from isocyanate terminated, star shaped poly(ethylene glycol-stat-propylene glycol) prepolymers (80% ethylene glycol, six arms, M(w) = 3000, 12,000, and 18,000; this compound will be referred to as "Star PEG" in the text). Due to the highly reactive endgroups, these molecules form a dense network on the substrate with a high polymer surface coverage. The two systems were compared regarding their ability to prevent unspecific adsorption of insulin and lysozyme. The layers were analyzed by ellipsometry, contact angle measurements, and XPS. Protein adsorption was monitored by surface MALDI-TOF MS and fluorescence microscopy. No protein adsorption could be detected on Star PEG coatings and on mPEG-ald 5000, whereas mPEG-ald 30,000 could only prevent adsorption of lysozyme but not of the smaller insulin.

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz).

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.

Immediate toxicity during fractionated total body irradiation as conditioning for bone marrow transplantation.

BACKGROUND: Total body irradiation followed by bone marrow transplantation is well established as a part of the conditioning regimen in high dose therapy. The immediate tolerance of fractionated total body irradiation (FTBI) was investigated prospectively. METHODS: From January 1995 to December 1998 162 patients received a FTBI, 6x2 Gy on 3 consecutive days, lung dose 10 Gy, for allogeneic (n=112) or autologous (n=50) bone marrow transplantation. High dose chemotherapy (mostly Cyclophosphamide) was administered after the FTBI. A standardized supportive therapy was administered. The immediate toxicity of FTBI was evaluated prospectively prior to each radiation fraction using a defined questionnaire. RESULTS: Main symptoms distressing the patient during irradiation period were gastrointestinal symptoms like nausea and emesis. The prevalence of nausea per fraction increased to 26.1% after the 4th fraction, with a significant higher prevalence in children younger than 10 years at 1st and 2nd fractions. 42.6 and 22. 8%, respectively, of all patients complained of nausea and episodes of emesis, during FTBI. Mild xerostomia and parotiditis were observed in 29.9 and 7.1% of all patients. Further gastrointestinal side effects during FTBI were loss of appetite in 16.0%, indisposition in 25.3%, mild oesophagitis in 3.7% and diarrhoea in 3. 7% of the patients. During FTBI 41.4% of the patients developed a temporary skin irritation (mild erythema). Pruritus was registered in 3.7% of the patients. Headache was observed in 14.8% and Fatigue syndrome in 49.2% of women and 28.3% of men (P<0.005). CONCLUSION: FTBI is a well tolerated therapeutic regimen in high dose therapy. The 162 patients investigated revealed no severe immediate side effects.

DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.

The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and primase.

The effect of positioning on the hand function of boys with cerebral palsy.

The effect of positioning (sitting and prone standing) on the hand function of 10 boys (mean age = 12.5 years, SD = 1.2 years) with spastic diplegic cerebral palsy was studied. Two groups of subjects were tested twice (Tests 1 and 2) with the Jebsen-Taylor Hand Function Test (Jebsen, Taylor, Treischmann, Trotter, & Howard, 1969; Taylor, Sand, & Jebsen, 1973) to measure rate of manipulation. In addition, a scale modified from Hohlstein (1982) was used to measure quality of grasp on each subtest of the Jebsen-Taylor test. No significant differences between the mean scores of the two groups were found on the total scores of the Jebsen-Taylor test, either between Tests 1 and 2 or between sitting and prone standing. When the data from Tests 1 and 2 were combined, it was found that on one subtest--simulated feeding--the subjects performed significantly faster while in a prone standing position. On another subtest--picking up small objects--the subjects performed significantly faster while in a sitting position. Except during the simulated feeding subtest, the quality of the subjects' grasp was observed to be mature and tailored to the objects manipulated. This paper presents considerations for analyzing positioning in relation to upper extremity tasks.

Atrial natriuretic peptide plasma levels during hemodialysis and hemofiltration in patients with end-stage renal disease.

Using a cross-over protocol we repeatedly measured the plasma levels of alpha-hANP (atrial natriuretic peptide) during one week by radio-immunoassay in eight patients with end-stage renal disease treated with chronic hemodialysis or hemofiltration. Before each hemodialysis or hemofiltration session mean plasma ANP levels (353 +/- 112, and 337 +/- 99 pg.ml-1, respectively) were significantly above normal (50 - 166 pg.ml-1). In all but one patient, the values fell significantly towards but not reaching the normal range. Plasma ANP concentrations returned to normal at the end of the treatment in only two of the eight subjects. There was a positive correlation between the increase in body weight from one treatment to the next and the plasma ANP concentration (r = +0.35, p less than 0.05). The net loss of fluid volume during each treatment did not correlate significantly with the change in plasma ANP levels. There was no difference between hemodialysis and hemofiltration. Plasma ANP measurement may be helpful in the judgement of volume status in patients with end-stage renal disease treated by hemodialysis or hemofiltration.

Beneficial effects of long-term prostaglandin E2 infusion on the course of postischemic acute renal failure. Long-term studies in chronically instrumented conscious dogs.

The effect of a continuous intra-aortal infusion of prostaglandin E2 (PGE2) (0.03 microgram . min-1 . kg-1) on the course of postischemic renal failure (180 min cessation of blood flow by inflation of a pneumatic cuff) has been investigated in 11 conscious sodium-replete dogs. The glomerular filtration rate (51Cr-EDTA: endogenous creatinine clearance) was less decreased in the PGE2 group (group B, n = 6) than in the control group (group A, n = 5; 13 ml . min-1 vs. 22 ml . min-1; p less than 0.05). Renal blood flow (electromagnetic flow probe) was markedly lower in the control group (82 ml . min-1) than in the PGE2 group (130 ml . min-1; p less than 0.05), even exceeding baseline levels in the latter group. Accordingly, the excessive rise in renal vascular resistance in the control group (+277%) was abolished in the PGE2 group (-20%) (p less than 0.05). Nitrogen retention was also markedly improved. Plasma renin activity, which was markedly raised initially (25.8 ng angiotensin I . ml-1 . h-1) was not significantly further increased during the subsequent 7 days. Urinary excretion of PGE2 was diminished in the control group and elevated following PGE2 infusion. It is suggested that the beneficial effects of PGE2 are mediated by preservation of renal perfusion. Additional effects of prostanoids on the ultrafiltration coefficient (KF) and cytoprotection by reduction of intracellular calcium accumulation must also be taken into consideration.