The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules.

Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.

Autologous chondrocyte implantation versus ACI using 3D-bioresorbable graft for the treatment of large full-thickness cartilage lesions of the knee.

BACKGROUND: In autologous chondrocyte implantation (ACI), the periosteum patch which is sutured over the cartilage defect has been identified as a major source of complications such as periosteal hypertrophy. In the present retrospective study, we compared midterm results of first-generation ACI with a periosteal patch to second generation ACI using a biodegradable collagen fleece (BioSeed-C) in 82 patients suffering from chronic posttraumatic and degenerative cartilage lesions of the knee. METHODS: Clinical outcome was assessed in 42 patients of group 1 and in 40 patients of group 2 before implantation of the autologous chondrocytes and at a minimum follow-up of 2 years using the ICRS score, the modified Cincinnati score and the Lysholm score. RESULTS: Although patients treated with BioSeed-C had more previous surgical procedures on their respective knees, highly significant improvements (P < 0.001) were assessed in both groups at comparable outcome levels: the ICRS score improved from grade D (poor) preoperatively to grade C (fair); the modified Cincinnati knee score from 3.26 to 6.4 (group 1) and 3.3 and 6.88 (group 2). Lysholm score improved from 33 to 70 points (group 1) and from 47 to 78 points (group 2), respectively. Revision surgery was due to symptomatic periosteal hypertrophy (n = 4), graft failure (n = 3), plica syndrome (n = 2) synovectomy (n = 1) (group 1); and graft failure (n = 2), debridement (n = 1), synovectomy (n = 2) (group 2). CONCLUSION: These results suggest that BioSeed-C is an equally effective treatment option for focal degenerative chondral lesions of the knee in this challenging and complex patient profile.

Porous tantalum and poly-epsilon-caprolactone biocomposites for osteochondral defect repair: preliminary studies in rabbits.

Currently, various techniques are in use for the repair of osteochondral defects, none of them being truly satisfactory and they are often two step procedures. Comorbidity due to cancellous bone harvest from the iliac crest further complicates the procedure. Our previous in vitro studies suggest that porous tantalum (TM) or poly-epsilon-caprolactone scaffolds (PCL) in combination with periosteal grafts could be used for osteochondral defect repair. In this in vivo study, cylindrical osteochondral defects were created on the medial and lateral condyles of 10 rabbits and filled with TM/periosteum or PCL/periosteum biosynthetic composites (n = 8 each). The regenerated osteochondral tissue was then analyzed histologically, and evaluated in an independent and blinded manner by five different observers using a 30-point histological score. The overall histological score for PCL/periosteum was significantly better than for TM/periosteum. However, most of the regenerates were well integrated with the surrounding bone (PCL/periosteum, n = 6.4; TM/periosteum, n = 7) along with partial restoration of the tidemark (PCL/periosteum, n = 4.4; TM/periosteum, n = 5.6). A cover of hyaline-like morphology was found after PCL/periosteum treatment (n = 4.8), yet the cartilage yields were inconsistent. In conclusion, the applied TM and PCL scaffolds promoted excellent subchondral bone regeneration. Neo-cartilage formation from periosteum supported by a scaffold was inconsistent. This is the first study to show in vivo results of both PCL and TM scaffolds for a novel approach to osteochondral defect repair.

Cell-laden and cell-free biopolymer hydrogel for the treatment of osteochondral defects in a sheep model.

The objective of the current study was to determine the suitability of cell-laden and cell-free alginate-gelatin biopolymer hydrogel for osteochondral restoration in a sheep model (n = 12). Four femoral defects per animal were filled with hydrogel (cHG) plus autologous chondrocytes (cHG + C) or periosteal cells (cHG + P) or gel only (cHG) or were left untreated (E). In situ solidification enabled instantaneous implant fixation. Sixteen weeks postoperatively, defect sites were processed for light microscopy and immunofluorescence. A modified Mankin and a semi-quantitative immunoreactivity score were used to evaluate histology and immunofluorescence, respectively. Defects after cHG + C were restored with smooth, hyaline-like neo-cartilage and trabecular subchondral bone. cHG + P and cHG treatments revealed slightly inferior regenerate morphology. Undifferentiated tissue was found in E. The histological score showed significant (p < 0.05) differences between all treatment groups. In conclusion, cHG induces satisfactory defect regeneration. Complete filling of the cavity in one step and subsequent rapid in situ solidification was feasible and facilitated graft fixation. Cell implantation might be beneficial, because cells seem to play a key role in histological outcome. Still, their contribution to the repair process remains unresolved because host cell influx takes place. The combination of alginate and gelatin, however, creates an environment capable of serving implanted and host cells for osteo-chondrogenic tissue regeneration.

Morphology and function of ovine articular cartilage chondrocytes in 3-d hydrogel culture.

Different cell- and biomaterial-based tissue engineering techniques are under investigation to restore damaged tissue. Strategies that use chondrogenic cells or tissues in combination with bioresorbable delivery materials are considered to be suitable to regenerate bio-artificial cartilage. Three-dimensional (3-D) cell embedding techniques can provide anchorage-independent cell growth and homogenous spatial cell arrangement, which play a key role in the maintenance of the characteristic phenotype and thus the formation of differentiated tissue. We developed a new injectable high water content (90%) hydrogel formulation with 5% sodium alginic acid and 5% gelatin as a temporary supportive intercellular matrix for 3-D cell culture. The objective was to determine whether the in vitro hydrogel culture of chondrocytes could preserve hyaline characteristics and thus could provide cartilage regeneration in vitro. Chondrocytes harvested from knee joints of skeletally mature sheep were cultured 3-D in hydrogel (7 x 10(6) cells/ml, 2.8-mul beads) for up to 10 weeks. Cell morphology and viability were evaluated with light microscopy, and proliferative activity was assessed with antibromodeoxyuridine immunofluorescence. Expression of collagens type I (COL1) and II (COL2), cartilage proteoglycans (PG) and hyaluronan synthases (HAS) were studied immunohistochemically. We observed that up to 36% of chondrocytes proliferated, while almost 100% presented a differentiated spheroidal phenotype. After an initial decrease at 2 weeks, cell density recovered to 85% of the initial absolute value at 10 weeks. Expression of hyaline matrix molecules resembled the in vivo pattern with increasing spatial deposition of PG and COL2. The proportion of PG-positive cells increased from initially 13 to 53% after 10 weeks, in contrast to consistently 100% COL2-positive cells. We conclude that 3-D hydrogel culture, even without mechanical stimulation or growth factor application, can keep chondrocytes in a differentiated state and provides a chondrogenic cell environment for in vitro cartilage regeneration for at least 10 weeks. Moreover, this hydrogel appears to be a suitable cell delivery material for subsequent in vivo implantation.

Sildenafil increased exercise capacity during hypoxia at low altitudes and at Mount Everest base camp: a randomized, double-blind, placebo-controlled crossover trial.

BACKGROUND: Alveolar hypoxia causes pulmonary hypertension and enhanced right ventricular afterload, which may impair exercise tolerance. The phosphodiesterase-5 inhibitor sildenafil has been reported to cause pulmonary vasodilatation. OBJECTIVE: To investigate the effects of sildenafil on exercise capacity under conditions of hypoxic pulmonary hypertension. DESIGN: Randomized, double-blind, placebo-controlled crossover study. SETTING: University Hospital Giessen, Giessen, Germany, and the base camp on Mount Everest. PARTICIPANTS: 14 healthy mountaineers and trekkers. MEASUREMENTS: Systolic pulmonary artery pressure, cardiac output, and peripheral arterial oxygen saturation at rest and during assessment of maximum exercise capacity on cycle ergometry 1) while breathing a hypoxic gas mixture with 10% fraction of inspired oxygen at low altitude (Giessen) and 2) at high altitude (the Mount Everest base camp). INTERVENTION: Oral sildenafil, 50 mg, or placebo. RESULTS: At low altitude, acute hypoxia reduced arterial oxygen saturation to 72.0% (95% CI, 66.5% to 77.5%) at rest and 60.8% (CI, 56.0% to 64.5%) at maximum exercise capacity. Systolic pulmonary artery pressure increased from 30.5 mm Hg (CI, 26.0 to 35.0 mm Hg) at rest to 42.9 mm Hg (CI, 35.6 to 53.5 mm Hg) during exercise in participants taking placebo. Sildenafil, 50 mg, significantly increased arterial oxygen saturation during exercise (P = 0.005) and reduced systolic pulmonary artery pressure at rest (P < 0.001) and during exercise (P = 0.031). Of note, sildenafil increased maximum workload (172.5 W [CI, 147.5 to 200.0 W]) vs. 130.6 W [CI, 108.8 to 150.0 W]); P < 0.001) and maximum cardiac output (P < 0.001) compared with placebo. At high altitude, sildenafil had no effect on arterial oxygen saturation at rest and during exercise compared with placebo. However, sildenafil reduced systolic pulmonary artery pressure at rest (P = 0.003) and during exercise (P = 0.021) and increased maximum workload (P = 0.002) and cardiac output (P = 0.015). At high altitude, sildenafil exacerbated existing headache in 2 participants. LIMITATIONS: The study did not examine the effects of sildenafil on normoxic exercise tolerance. CONCLUSIONS: Sildenafil reduces hypoxic pulmonary hypertension at rest and during exercise while maintaining gas exchange and systemic blood pressure. To the authors' knowledge, sildenafil is the first drug shown to increase exercise capacity during severe hypoxia both at sea level and at high altitude.