One-step surgical procedure for the treatment of osteochondral defects with adipose-derived stem cells in a caprine knee defect: a pilot study.

Regenerative therapies offer attractive alternatives for the treatment of osteochondral defects. Adipose-derived stromal vascular fraction (SVF) cells allow the development of one-step surgical procedures by their abundant availability and high frequency. In this pilot study we evaluated the in vivo safety, feasibility, and efficacy of this concept using scaffolds seeded with freshly isolated (SVF) or cultured adipose stem cells (ASCs), and compared these to their acellular counterparts. Osteochondral defects were created in medial condyles and trochlear grooves in knees of eight goats. Defects were filled with acellular collagen I/III scaffolds or scaffolds seeded with SVF cells or cultured ASCs. Osteochondral regeneration was evaluated after 1 and 4 months by macroscopy, immunohistochemistry, biomechanical analysis, microCT analysis, and biochemistry. After 1 month, no adverse effects were noted. Microscopic, but not macroscopic evaluation showed considerable yet not significant differences, with cell-loaded constructs showing more extensive regeneration. After 4 months, acellular constructs displayed increased regeneration, however, to a lesser degree than cell-treated constructs. The latter exhibited more extensive collagen type II, hyaline-like cartilage, and higher elastic moduli, and their glycosaminoglycan content in the cartilaginous layer better approached native tissue values. Moreover, their defect regions contained higher levels of regenerated, mature subchondral bone with more intense collagen type I staining. SVF cells tended to perform best on all parameters. In summary, this pilot study demonstrated the preclinical safety and feasibility of a one-step surgical procedure for osteochondral defect regeneration. Similar regeneration was found between freshly isolated SVF cells and cultured ASCs. Larger studies with longer follow-up are required to substantiate these findings.

Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel.

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFß1-induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4 days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFß1-induced group. At 21 days, the TGFß1-treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21 days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link-p gene expression compared with the non-induced group, and to similar levels as the TGFß1-induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three-dimensional (3D) micro-environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFß1. This might lead to an interesting alternative when considering short-term triggering in a one-step surgical procedure for the treatment of cartilaginous defects.

Freshly isolated stromal cells from the infrapatellar fat pad are suitable for a one-step surgical procedure to regenerate cartilage tissue.

BACKGROUND AIMS: Stem cell therapies are being evaluated as promising alternatives for cartilage regeneration. We investigated whether stromal vascular fraction cells (SVF) from the infrapatellar (Hoffa) fat pad are suitable for a one-step surgical procedure to treat focal cartilage defects. METHODS: SVF was harvested from patients undergoing knee arthroplasty (n = 53). Colony-forming unit (CFU) assays, growth kinetics and surface marker profiles were determined, and the chondrogenic differentiation capacity of freshly isolated SVF was assessed after seeding in three-dimensional poly (L-lactic-co-epsilon-caprolactone) scaffolds. RESULTS: SVF yield per fat pad varied between 0.55 and 16 x 10(6) cells. CFU frequency and population doubling time were 2.6 +/- 0.6% and +/-2 days, respectively. Surface marker profiles matched those of subcutaneous-derived adipose-derived stem cells (ASC). CFU from Hoffa SVF showed differentiation toward osteogenic and adipogenic lineages. Cartilage differentiation was confirmed by up-regulation of the cartilage genes sox9, aggrecan, collagen type II and cartilage oligomeric matrix protein (COMP), collagen II immunostaining, Alcian Blue staining and glycosaminoglycan production. Compared with passaged cells, SVF showed at least similar chondrogenic potential. CONCLUSIONS: This study demonstrates that SVF cells from the infrapatellar fat pad are suitable for future application in a one-step surgical procedure to regenerate cartilage tissue. SVF shows similar favorable characteristics as cultured ASC, and chondrogenic differentiation even appears to be slightly better. However, because of variable harvesting volumes and yields, SVF from the infrapatellar fat pad might only be applicable for treatment of small focal cartilage defects, whereas for larger osteoarthritic defects subcutaneous adipose tissue depot would be preferable.

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies.

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 +/- 1.1%, mean +/- SEM) and hip/thigh region (1.2 +/- 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells.

Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue engineering purposes, however, it would be advantageous to use the whole SVF, which can be transplanted without further in vitro selection or expansion steps. Because little is known about the freshly isolated ASCs in the SVF, we phenotypically characterized human freshly isolated ASCs, using flow cytometry. In addition, we investigated whether freshly isolated ASCs have functional properties comparable to cultured ASCs. For this, the differentiation potential of both freshly isolated ASCs and cultured ASCs into the osteogenic pathway was analyzed. Freshly isolated ASCs slightly differed in immunophenotype from cultured ASCs. Contrary to cultured ASCs, freshly isolated ASCs were shown to be highly positive for CD34, and positive for CD117 and HLA-DR. On the other hand, expression of CD105 and especially CD166 on the freshly isolated ASCs was relatively low. After osteogenic stimulation of freshly isolated ASCs, both Runx-2 and CollaI gene expression were significantly increased (p < 0.05). However, there was a difference in the kinetics of gene expression between freshly isolated and cultured ASCs and also between the different SVF isolates tested. There was no difference in alkaline phosphatase activity between freshly isolated ASCs and cultured ASCs. In addition, freshly isolated ASCs stained positive for osteonectin and showed matrix mineralization. We conclude that although there are minor differences in phenotype and kinetics of differentiation between freshly isolated ASCs and cultured ASCs, the use of freshly isolated ASCs for tissue engineering purposes involving bone repair is potentially applicable.

Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles.

In this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20wt% poly (dl-lactic-co-glycolic acid) (PLGA) microparticles were investigated in a rat cranial defect. After 2, 4 and 8 weeks of implantation, implants were evaluated mechanically (push-out test) and morphologically (Scanning Electron Microscopy (SEM) and histology). The results of the push-out test showed that after 2 weeks the shear strength of the implants was 0.44+/-0.44MPa (average+/-sd), which increased to 1.34+/-1.05MPa at 4 weeks and finally resulted in 2.60+/-2.78MPa at 8 weeks. SEM examination showed a fracture plane at the bone-cement interface at 2 weeks, while the 4- and 8-week specimens created a fracture plane into the CaP/PLGA composites, indicating an increased strength of the bone-cement interface. Histological evaluation revealed that the two weeks implantation period resulted in minimal bone ingrowth, while at 4 weeks of implantation the peripheral PLGA microparticles were degraded and replaced by deposition of newly formed bone. Finally, after 8 weeks of implantation the degradation of the PLGA microparticles was almost completed, which was observed by the bone ingrowth throughout the CaP/PLGA composites. On basis of our results, we conclude that the shear strength of the bone-cement interface increased over time due to bone ingrowth into the CaP/PLGA composites. Although the bone-cement contact could be optimized with an injectable CaP cement to enhance bone ingrowth, still the mechanical properties of the composites after 8 weeks of implantation are insufficient for load-bearing purposes.