Intermittent Hypoxic Therapy Inhibits Allogenic Bone-Graft Resorption by Inhibition of Osteoclastogenesis in a Mouse Model.

Systemic Intermittent Hypoxic Therapy (IHT) relies on the adaptive response to hypoxic stress. We investigated allogenic bone-graft resorption in the lumbar spine in 48 mice. The mice were exposed to IHT for 1 week before surgery or 1 week after surgery and compared with controls after 1 and 4 weeks. Complete graft resorption was observed in 33-36% of the animals in the control group, but none in the preoperative IHT group. Increased bone-graft volume was demonstrated by micro-computed tomography in the preoperative IHT group after 1 week (p = 0.03) while a non-significant difference was observed after 4 weeks (p = 0.12). There were no significant differences in the postoperative IHT group. Increased concentration of immune cells was localized in the graft area, and more positive tartrate-resistant acid phosphatase (TRAP) staining was found in controls compared with IHT allogenic bone grafts. Systemic IHT resulted in a significant increase of the major osteoclast inhibitor osteoprotegerin as well as osteogenic and angiogenic regulators Tgfbr3, Fst3l, Wisp1, and Vegfd. Inflammatory cytokines and receptor activator of nuclear factor kappa-B ligand (RANKL) stimulators IL-6, IL-17a, IL-17f, and IL-23r increased after 1 and 4 weeks, and serum RANKL expression remained constant while Ccl3 and Ccl5 decreased. We conclude that the adaptive response to IHT activates numerous pathways leading to inhibition of osteoclastic activity and inhibition of allogenic bone-graft resorption.

No effect of platelet-rich plasma as adjuvant to bone marrow stimulation for the treatment of chondral defects in a large animal model.

BACKGROUND: Bone marrow stimulation (BMS) remains a dominant treatment strategy for symptomatic full thickness articular cartilage defects. Autologous platelet-rich plasma (PRP), may improve biological cartilage repair as an adjunct to BMS. OBJECTIVES: To assess the histological quality of cartilage repair after BMS with and without repeated local injections of PRP for the treatment of full-thickness focal chondral defects of the knee. METHODS: Two full-thickness chondral defects (Ø = 6 mm) were surgically performed in the medial and lateral trochlea of each knee in skeletally mature Göttingen minipigs. The two treatment groups with 12 defect for each groups were (1) BMS with one weekly PRP injection for 4 weeks, and (2) BMS alone. The animals were euthanized after 6 months. Samples of both whole blood and PRP were analysed with an automated hematology analyzer to determine the concentrations of platelets and nucleated cells. The composition of cartilage repair tissue was assessed using gross appearance assessment, histomorphometry and semi-quantitative scoring (ICRS II). RESULTS: The average fold increase in platelets was 10.2 ± 2.2. Leukocyte concentration increased in PRP samples by an average fold change of 7.2 ± 1.3. Our macroscopic findings showed that the defects in the BMS + PRP-treated group, were filled with an irregular, partially rough tissue similar to the BMS-treated group. No significant difference in amount of hyalin cartilage, fibrocartilage or fibrous tissue content and ICRS II scores was found between the groups. CONCLUSIONS: Four repeated local injections of leukocyte-rich PRP after BMS in the treatment of full-thickness cartilage injuries demonstrated no beneficial effects in terms of macroscopic and histological cartilage repair tissue quality.

Poor osteochondral repair by a biomimetic collagen scaffold: 1- to 3-year clinical and radiological follow-up.

INTRODUCTION: Treatment of osteochondral injuries is challenging, and no gold standard has been established. Layered cell-free scaffolds are a new treatment option for these defects. The aim of this study was to evaluate the osteochondral repair in patients treated with the MaioRegen(®) scaffold, a cell-free biomimetic scaffold consisting of type I collagen and hydroxyapatite. Treatment using this scaffold has previously shown promising clinical results. METHODS: Ten patients with osteochondral lesions in the knee (n = 6) or in the talus (n = 4) were enrolled. The patients underwent pre-operative MRI and CT scans and were assessed at 1- and 2.5-year timescales post-operatively. The cartilage and bone formations were evaluated semi-quantitatively using the MOCART score. Knee patients were clinically evaluated using KOOS, subjective IKDC and Tegner scores, whereas ankle patients were evaluated using AOFAS Hindfoot and Tegner scores. RESULTS: Two patients were re-operated and excluded from further follow-up due to treatment failure. None of the patients had complete regeneration of the subchondral bone evaluated using CT. At 2.5 years, 6/8 patients had no or very limited (<10 %) bone formation in the defects and 2/8 had 50-75 % bone formation in the treated defect. MRI showed no improvement in the MOCART score at any time point. The IKDC score improved from 41.3 to 80.7, and the KOOS pain subscale improved from 63.8 to 90.8 at 2.5-year follow-up. No improvement was found with the remaining KOOS subscales, the Tegner or AOFAS Ankle-Hindfoot score. CONCLUSION: Treatment of osteochondral defects in the ankle and knee joint with a biomimetic scaffold resulted in incomplete cartilage repair and poor subchondral bone repair at 1- and 2.5-year follow-up. Clinical significant improvements were observed. These results raise serious concerns about the biological repair potential of the MaioRegen(®) scaffold, and we advise to use the MaioRegen(®) scaffold with caution. LEVEL OF EVIDENCE: Prospective therapeutic study, Level IV.

Cellular senescence in aging and osteoarthritis.

- It is well accepted that age is an important contributing factor to poor cartilage repair following injury, and to the development of osteoarthritis. Cellular senescence, the loss of the ability of cells to divide, has been noted as the major factor contributing to age-related changes in cartilage homeostasis, function, and response to injury. The underlying mechanisms of cellular senescence, while not fully understood, have been associated with telomere erosion, DNA damage, oxidative stress, and inflammation. In this review, we discuss the causes and consequences of cellular senescence, and the associated biological challenges in cartilage repair. In addition, we present novel strategies for modulation of cellular senescence that may help to improve cartilage regeneration in an aging population.

Hypoxia enhances chondrogenic differentiation of human adipose tissue-derived stromal cells in scaffold-free and scaffold systems.

Human adipose-derived stromal cells (hASCs) possess the potential for chondrogenic differentiation. Recent studies imply that this differentiation process may be enhanced by culturing the cells in low oxygen tension in combination with three-dimensional (3D) scaffolds. We report the evaluation of the chondrogenic potential of hASC pellets in 5 and 21% O2 and as cell-scaffold constructs using a collagen I/III scaffold with chemical induction using TGF-ß3. hASCs from four human donors were cultured both in a micromass pellet system and in 3D collagen I/III scaffolds in either 5 or 21% O2. Chondrogenesis was evaluated by quantitative gene expression analysis of aggrecan, SOX9, collagen I, II and X and histological evaluation with H&E and toluidine blue staining. Induced pellets cultured in 5% O2 showed increased peripheral cellularity and matrix deposition compared with 21% O2. Induced pellets cultured in 5% O2 had increased control-adjusted gene expression of aggrecan, SOX9 and collagen I and decreased collagen X compared with 21% O2 cultures. Induced pellets had higher gene expression of aggrecan, SOX9, collagen I, II and X and increased ratios of collagen II/I and collagen II/X compared with controls. As for pellets, scaffold cultures showed cellularity and matrix deposition organized in a zonal manner as a function of the oxygen tension, with a cartilage-like morphology and matrix deposition peripherally in the 5% O2 group and a more centrally located matrix in the 21% O2 group. There were no differences in histology and gene expressions between pellet and scaffold cultures. Five percent O2 in combination with chondrogenic culture medium stimulated chondrogenic differentiation of hASCs in vitro. We observed similar patterns of differentiation and matrix disposition in pellet and scaffold cultures.

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

PURPOSE: This study investigates the effect of cell seeding density on cartilage repair in matrix-assisted chondrocyte implantation in vitro and in vivo. METHODS: In vitro: Four different cell seeding densities of human chondrocytes were seeded onto a porous methoxy-polyethylene glycol-polylactic-co-glycolic acid scaffold (MPEG-PLGA) polymer scaffold ASEED™ (1.2 × 10(6), 4.0 × 10(6), 1.2 × 10(7) and 2.0 × 10(7) cells/cm(3)). The cartilage repair response was evaluated by relative gene expression of the chondrogenic markers sox9, collagen types I, II and X, and aggrecan, total DNA content and sulphated glycosaminoglycan synthesis. In vivo: Using a New Zealand white rabbit intercondylar osteochondral defect model, three different cell seeding densities (1.2 × 10(6), 4.0 × 10(6) and 1.2 × 10(7) cells/cm(3)) were tested with an empty scaffold as control. The cartilage repair response was evaluated using O'Driscoll score. RESULTS: In vitro: A significant difference (p < 0.05) in total DNA content was found at day 2 but not at day 7. The low cell seeding densities yielded the highest GAG content (p < 0.001) at day 7. Collagen type I was highest (p < 0.01) at the lowest density at day 7. In vivo: No significant difference was found between the 4 groups. CONCLUSIONS: No positive effect on cartilage repair was found using increased cell seeding density. LEVEL OF EVIDENCE: Controlled experimental study, Level II.

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

PURPOSE: To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide) scaffold. METHODS: By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water-dioxane-PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT-PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O'Driscoll score. RESULTS: In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells. CONCLUSION: The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide scaffold with cells.

Dermatan sulphate in methoxy polyethylene glycol-polylactide-co-glycolic acid scaffolds upregulates fibronectin gene expression but has no effect on in vivo osteochondral repair.

PURPOSE: The purpose of the study was to investigate the effect of dermatan sulphate (DS) addition to biodegradable methoxy polyethylene glycol (MPEG) substituted polylactide-co-glycolic acid (PLGA) scaffolds for cartilage repair in vitro and in vivo. METHODS: Human chondrocytes from eight patients undergoing anterior cruciate ligament reconstruction were isolated and cultured in 5% oxygen on MPEG-PLGA scaffolds±DS for one, three, seven and 14 days. Analyses were performed using quantitative gene expression analysis for chondrogenic and cell attachment markers. An osteochondral drill hole defect was created in the intertrochlear groove of the distal femur in 20 New Zealand white rabbits (defects n=20). When bleeding was observed, the defects were treated with MPEG-PLGA scaffolds±DS. Twelve weeks after surgery the rabbits were sacrificed and the defects were analysed using histological grading with O'Driscoll scoring. RESULTS: DS addition to MPEG-PLGA scaffolds resulted in a significant upregulation of fibronectin gene expression on day 1. No differences were observed in chondrogenic gene expression. There were no differences between the two groups in histological grading (+DS 10.3 and -DS 9.6). CONCLUSIONS: Upregulation of fibronectin in vitro indicating early cell-scaffold interaction and attachment did not result in improved cartilage repair in an osteochondral defect model in rabbits.

Chondrocyte gene expression is affected by very small iron oxide particles-labeling in long-term in vitro MRI tracking.

PURPOSE: To investigate the effect and dose response of very small iron oxide particles (VSOP) labeling of human chondrocytes for long-term in vitro MRI tracking. MATERIALS AND METHODS: Chondrocytes were isolated from cartilage biopsies from four patients. The cells for the dose-response study were labeled with 25, 50, or 100 µg/mL VSOP. Quantitative gene expression and cellular proliferation were compared with unlabeled controls at day 1, 3, and 7. The cells suited for MRI tracking were labeled with 50 µg/mL VSOP and embedded in alginate beads, followed by MRI (using T2-weighted sequences) at day 0, 1, 3, 7, 14, 21, 28, and histology was performed at each time-point. RESULTS: Histology revealed that VSOP particles were intracellularly confined at all time-points, whereas no extracellular VSOPs were observed. A mean reduction in T2-value of 25.1 ms (±SD 3.5 ms) was found on T2-maps. The chondrocyte-specific genes aggrecan, collagen type 2, and sox9 were all affected by labeling, the two latter in a dose-dependent manner. VSOPs had no effect on proliferation. CONCLUSION: VSOP labeling of chondrocytes affected gene expression but not proliferation. The labeled chondrocytes could be recognized by MRI for 4 weeks without significant changes in the T2 relaxation time.

Demographics in Patients Receiving Matrix-Assisted Chondrocyte Implantation (MACI) in the Ankle.

Objective. To compare demographics and cartilage lesion characteristics of patients enrolled in clinical trials investigating autologous chondrocyte implantation (ACI) in the ankle joint with those actually scheduled for matrix-assisted chondrocyte implantation (MACI) using database records. Design. Anonymized data from patients scheduled for MACI treatment in the ankle in Australia/Asia and Europe were obtained from the Genzyme/Sanofi database. Average age, defect size, and male-female ratio were analyzed and compared by country. A literature search was performed on PubMed and Google Scholar and clinical cohort studies and prospective comparative trials using ACI and related treatments in the ankle joint were identified. Weighted average age, weighted defect size, and male-female ratio were analyzed and compared with database data. Results. The 167 patients included from the databases from Europe and Australia had a mean age of 33.4 years (range 14-64 years) and a mean defect size of 2.27 cm2 (range 0.25-16 cm2). Male-female ratio was 4:3. Patients from European countries were significantly younger and had significantly larger defects compared with patients from Australia. From the literature search a total of 472 patients were included from 28 studies. The mean age was 32.2 years (range 15-62 years). Male-female ratio was 3:2. Weighted mean size was 1.94cm2 (range 0.3-16). There were no significant differences between previous studies and databases. Conclusion. No differences in sizes and age were found between patients enrolled in clinical trials and patients scheduled for MACI outside clinical trials. The sizes of treated defects followed the general recommendations. There were, however, significant differences between countries.

The Effect of Normobaric Intermittent Hypoxia Therapy on Bone in Normal and Disuse Osteopenic Mice.

Bromer, Frederik Duch, Mikkel Bo Brent, Michael Pedersen, Jesper Skovhus Thomsen, Annemarie Brüel, and Casper Bindzus Foldager. The effect of normobaric intermittent hypoxia therapy on bone in normal and disuse osteopenic mice. High Alt Med Biol. 22: 225-234, 2021. Background: Systemic intermittent hypoxia therapy (IHT) has been shown to elicit beneficial effects on multiple physiological systems. However, only few studies have investigated the effect of long-term normobaric IHT on bone mass and mechanical and microstructural properties. The aim of the present study was to examine the effect of IHT on bone in both healthy and osteopenic mice. Materials and Methods: Thirty mice were stratified into four groups: Ctrl, Ctrl+IHT, Botox, and Botox+IHT. Osteopenia was induced by injecting Botox into the right hindlimb of the mice causing paralysis and disuse. IHT animals were placed in a normobaric hypoxia-chamber (10% oxygen) for 1 hour twice daily 5 days/week. Animals were sacrificed after 21 days, and DEXA, micro-computed tomography, and mechanical testing were performed on the femora. Results: As expected, Botox resulted in a significant reduction of bone mineral content (-23.4%), area bone mineral density (-19.1%), femoral neck strength (Fmax: -54.7%), bone volume fraction (bone volume/tissue volume: -41.8%), and trabecular thickness (-32.4%). IHT had no measurable effect on the bone properties in either healthy or osteopenic mice. Conclusion: The study confirmed that Botox led to loss of bone mass, deterioration of trabecular microstructure, and loss of bone strength. These changes were not influenced by IHT. Notably, IHT had no detrimental effect on bone in either healthy or osteopenic mice. This indicates that IHT of ailments outside of the skeletal system may be administered without causing harm to the bone.

No Effect of Platelet-Rich Plasma Injections as an Adjuvant to Autologous Cartilage Chips Implantation for the Treatment of Chondral Defects.

BACKGROUND: Repair of chondral injuries using cartilage chips has recently demonstrated clinical feasibility. Autologous platelet-rich plasma (PRP) is a potential promising technique for improving healing response during cartilage repair. PURPOSE: To assess the cartilage repair tissue quality after autologous cartilage chips treatment (CC) with and without repeated local injections of PRP for the treatment of full-thickness focal chondral defects of the knee. MATERIALS AND METHODS: Two full-thickness chondral defects (Ø = 6 mm) were created in the medial and lateral trochlea facets of each knee in 6 skeletally mature Göttingen minipigs. The 2 treatment groups were (1) CC with 1 weekly PRP injection for 3 weeks (n = 12) and (2) CC alone (n = 12). The animals were euthanized after 6 months. Samples of whole blood and PRP were analyzed for concentrations of platelets and nucleated cells. The composition of the cartilage repair tissue was assessed using gross appearance assessment, histomorphometry, and semiquantitative scoring (ICRS II). RESULTS: Histological evaluation demonstrated no significant difference in the content of hyaline cartilage (CC + PRP: 18.7% vs. CC: 19.6%), fibrocartilage (CC + PRP: 48.1% vs. CC: 51.8%), or fibrous tissue (CC + PRP: 22.7% vs. CC: 21.8%) between the treatment groups. Macroscopic evaluation did not demonstrate any difference between groups. CONCLUSIONS: PRP injections after CC in the treatment of full-thickness cartilage injuries demonstrated no beneficial effects in terms of macroscopic and histologic composition of cartilage repair tissue.

Particulated Cartilage for Chondral and Osteochondral Repair: A Review.

INTRODUCTION: Injuries to articular cartilage have a poor spontaneous repair potential and no gold standard treatment exist. Particulated cartilage, both auto- and allograft, is a promising new treatment method that circumvents the high cost of scaffold- and cell-based treatments. MATERIALS AND METHODS: A comprehensive database search on particulated cartilage was performed. RESULTS: Fourteen animal studies have found particulated cartilage to be an effective treatment for cartilage injuries. Many studies suggest that juvenile cartilage has increased regenerative potential compared to adult cartilage. Sixteen clinical studies on 4 different treatment methods have been published. (1) CAIS, particulated autologous cartilage in a scaffold, (2) Denovo NT, juvenile human allograft cartilage embedded in fibrin glue, (3) autologous cartilage chips-with and without concomitant bone grafting, and (4) augmented autologous cartilage chips. CONCLUSION: Implantation of allogeneic and autologous particulated cartilage provides a low cost and effective treatment alternative to microfracture and autologous chondrocyte implantation. The methods are promising, but large randomized controlled studies are needed.

[Safe surgical dislocation of the hip to treatment of injuries after traumatic hip injury in children].

Traumatic dislocation of the hip in children is rare. Immediately closed reduction is important but contains a risk of tissue becoming intertwined in the joint space, and a risk of development of avascular femoral head necrosis. In this case report, we present two cases of traumatic hip luxation in an 11-year-old boy and a nine-year-old boy, where intertwined labral tears were recognised immediately following closed reduction in one case and after two weeks in the other. They were treated with safe surgical dislocation of the femoral head. Blood flow to the femoral head was ensured by perioperative monitoring, and both boys had complete recovery.

Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes.

BACKGROUND: Hypoxic culturing of chondrocytes is gaining increasing interest in cartilage research. Culturing of chondrocytes under low oxygen tension has shown several advantages, among them increased synthesis of extracellular matrix and increased redifferentiation of dedifferentiated chondrocytes. Quantitative gene expression analyses such as quantitative real-time PCR (qRT-PCR) are powerful tools in the investigation of underlying mechanisms of cell behavior and are used routinely for differentiation and phenotype assays. However, the genes used for normalization in normoxic cell-cultures might not be suitable in the hypoxic environment. The objective of this study was to determine hypoxia-stable housekeeping genes (HKG) for quantitative real-time PCR (qRT-PCR) in human chondrocytes cultured in 21%, 5% and 1% oxygen by geNorm and NormFinder analyses. RESULTS: The chondrocytic response to the hypoxic challange was validated by a significant increase in expression of the hypoxia-inducible gene ankyrin repeat 37 as well as SOX9 in hypoxia. When cultured on the 3-dimentional (3D) scaffold TATA-binding protein (TBP) exhibited the highest expression stability with NormFinder while Ribosomal protein L13a (RPL13A) and beta2-microglobulin (B2M) were the most stable using geNorm analysis. In monolayer RPL13A were the most stable gene using NormFinder, while geNorm assessed RPL13A and human RNA polymerase II (RPII) as most stable. When examining the combination of (3D) culturing and monolayer RPL13A and B2M showed the highest expression stability from geNorm analysis while RPL13A also showed the highest expression stability using NormFinder. Often used HKG such as beta actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the most unstable genes investigated in all comparisons. The pairwise variations for the two most stable HKG in each group were all below the cut-off value of 0.15, suggesting that the two most stable HKG from geNorm analysis would be sufficient for qRT-PCR. CONCLUSION: All data combined we recommend RPL13A, B2M and RPII as the best choice for qRT-PCR analyses when comparing normoxic and hypoxic cultured human chondrocytes although other genes might also be suitable. However, the matching of HKG to target genes by means of a thorough investigation of the stability in each study would always be preferable.

A Standardized Method of Applying Toluidine Blue Metachromatic Staining for Assessment of Chondrogenesis.

OBJECTIVES: Staining with toluidine blue is a well-established procedure for the histological assessment of cartilaginous- and chondrogenic-differentiated tissues. Being a cationic dye, toluidine blue staining visualizes proteoglycans in a tissue because of its high affinity for the sulfate groups in proteoglycans. It is generally accepted that metachromatic staining with toluidine blue represents cartilaginous matrix and that the degree of positive staining corresponds with the amount of proteoglycans. DESIGN: Articular cartilage and pellets of chondrocytes or bone marrow stromal cells were analyzed with a standardized staining procedure for toluidine blue. RESULTS: In the present study, we illustrate why such an assumption is invalid unless a detailed description of the procedure and/or reference to a detailed published method are provided. This is because the staining specificity and intensity depend, as we have shown, on the pH of the staining solution, the use of dehydration, and on staining time. CONCLUSIONS: We can, therefore, suggest a well-controlled standardized protocol for toluidine blue staining, which provides an easy and simple selective staining technique for the assessment of cartilage tissue and proteoglycan development in chondrogenic differentiation. If this procedure is not used, then investigators must provide sufficient technical information concerning the staining protocol to allow an assessment of the validity of the staining results.

Age-Dependent Systemic Effects of a Systemic Intermittent Hypoxic Therapy In Vivo.

Introduction: The adaptive response to systemic intermittent hypoxic therapy (SIHT) may be used for therapeutic advances due to the activation of multiple pathways involved in angiogenesis, immunomodulation, and tissue homeostasis. The aim of this study was to investigate the early age-dependent systemic response of different exposures of SIHT in mice. Materials and Methods: Sixty-four C57BL/6NRj female mice in three different age groups, young (4-5 weeks), adolescent (8-10 weeks), and adults (23-32 weeks), were exposed to SIHT. Different algorithms for equal hypoxic challenges (oxygen-decrease*time) were investigated to allow examination of the role of absolute hypoxia (oxygen-decrease) compared with relative hypoxia (total oxygen depletion over time). The systemic effects of angiogenetic regulation were investigated using blood samples analyzed by ELISA, proteome profiles, and proximity extension immunoassay. One-way analysis of variance with post hoc Bonferroni analyses was performed. Results: The early systemic response to SIHT was dependent on the absolute hypoxia rather than relative hypoxia over time. Serum erythropoietin (EPO) levels were increased significantly in young mice receiving low-oxygen SIHT treatments (10% and 15% oxygen). The expression of angiogenic proteins differed between the different age groups indicating an age-dependent response to SIHT. Focusing on hypoxia-inducible factor-1 (HIF-1) signaling, there was a trend toward upregulated angiogenetic response with younger age. Furthermore, clustering of protein expression in low-oxygen SIHT algorithms were found between young and adolescent mice. In adult mice, the majority of the proteins were downregulated as a response to SIHT. The systemic response of metabolites expressions was most pronounced in young mice. Systemic levels of cardiac troponin I (Tnni3) was unaffected by SIHT independent of age groups. Conclusions: The systemic response to SIHT is dependent on the absolute hypoxic exposure rather than the relative hypoxic depletion over time. Age-dependent effects of a short-term SIHT were associated with an increase in EPO, upregulation of angiogenetic pathways, and select metabolic and cell-surface proteins.

Non-invasive cell tracking of SPIO labeled cells in an intrinsic regenerative environment: The axolotl limb.

Non-invasive methods to track the progress of stem cell therapies are important in the development of future regenerative therapies. Super-paramagnetic iron oxide particles (SPIOs) have previously been applied to track cells using magnetic resonance imaging (MRI) in vivo in non-regenerative animal models. To the best of the author's knowledge, the present study investigated for the first time, the feasibility of tracking SPIO labeled cells in an intrinsic regenerative environment, the regenerating limb of the axolotl, and investigated the homing of stem cell-like blastema cells to the regenerative zone. Viability and labeling success of labeled axolotl blastema cells was tested in vitro using cell culture and histology. SPIO labeling was performed in situ by intramuscular injections and mapped using MRI. Enhanced permeability and retention (EPR) effects were evaluated in the blastema, liver, heart, kidney and a back muscle. Finally, SPIO/Fluorophore-labeled blastema cells were injected intravascularly and tracked using MRI and fluorescence imaging. It was demonstrated that SPIO labeling had no effect on axolotl cell viability in vitro. In situ labeling resulted in an MRI signal alteration during 48 days of regeneration. EPR effect of unbound SPIO was observed only in the liver. MRI tracking revealed increased concentrations of SPIO labeled blastema cells in the liver, kidney and heart, however not the blastema of intravascularly injected axolotls. In conclusion, the results demonstrated that SPIO labeling facilitated non-invasive tracking of injected cells in the regenerating axolotl limb. An early homing mechanism of injected blastema cells to an injury site was not observed.

Surface chemistry, substrate, and topography guide the behavior of human articular chondrocytes cultured in vitro.

Understanding the behavior of chondrocytes in contact with artificial culture surfaces is becoming increasingly important in attaining appropriate ex vivo culture conditions of chondrocytes in cartilage regeneration. Chondrocyte transplantation-based cartilage repair requires efficiently expanded chondrocytes, and the culture surface plays an important role in guiding the behavior of the cell. Micro- and nano-engineered surfaces make it possible to modulate cell behavior. We hypothesized that the combined influence of topography, substrate, and surface chemistry may affect the chondrocyte culturing in terms of proliferation and phenotypic means. Human chondrocytes were cultured on polystyrene fabricated microstructures, flat polydimethylsiloxane (PDMS), or polystyrene treated with fibronectin or oxygen plasma and cultured for 1, 4, 7, and 10 days. The behavior of chondrocytes was evaluated by proliferation, viability, chondrogenic gene expression, and cell morphology. Contrary to our hypothesis, microstructures in polystyrene did not significantly influence the behavior of chondrocytes neither under normoxic- nor hypoxic conditions. However, changes in the substrate stiffness and surface chemistry were found to influence cell viability, gene expression, and morphology of human chondrocytes. Oxygen plasma treatment was the most important parameter followed by the softer substrate type PDMS. The findings indicate the culture of human chondrocytes on softer substratum and surface activation by oxygen plasma may prevent dedifferentiation and may improve chondrocyte transplantation-based cartilage repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2805-2816, 2018.

Combinatorial Screening of Nanoclay-Reinforced Hydrogels: A Glimpse of the "Holy Grail" in Orthopedic Stem Cell Therapy?

Despite the promise of hydrogel-based stem cell therapies in orthopedics, a significant need still exists for the development of injectable microenvironments capable of utilizing the  regenerative potential of donor cells. Indeed, the quest for biomaterials that can direct stem cells into bone without the need of external factors has been the "Holy Grail" in orthopedic stem cell therapy for decades. To address this challenge, we have utilized a combinatorial approach to screen over 63 nanoengineered hydrogels made from alginate, hyaluronic acid, and two-dimensional nanoclays. Out of these combinations, we have identified a biomaterial that can promote osteogenesis in the absence of well-established differentiation factors such as bone morphogenetic protein 2 (BMP2) or dexamethasone. Notably, in our "hit" formulations we observed a 36-fold increase in alkaline phosphate (ALP) activity and a 11-fold increase in the formation of mineralized matrix, compared to the control hydrogel. This induced osteogenesis was further supported by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy. Additionally, the Montmorillonite-reinforced hydrogels exhibited high osteointegration as evident from the relatively stronger adhesion to the bone explants as compared to the control. Overall, our results demonstrate the capability of combinatorial and nanoengineered biomaterials to induce bone regeneration through osteoinduction of stem cells in a natural and differentiation-factor-free environment.