Benjamin D. Elder

Hydrostatic Pressure in Articular Cartilage Tissue Engineering: From Chondrocytes to Tissue Regeneration

Cartilage has a poor intrinsic healing response, and neither the innate healing response nor current clinical treatments can restore its function. Therefore, articular cartilage tissue engineering is a promising approach for the regeneration of damaged tissue. Because cartilage is exposed to mechanical forces during joint loading, many tissue engineering strategies use exogenous stimuli to enhance the biochemical or biomechanical properties of the engineered tissue. Hydrostatic pressure (HP) is emerging as arguably one of the most important mechanical stimuli for cartilage, although no optimal treatment has been established across all culture systems. Therefore, this review evaluates prior studies on articular cartilage involving the use of HP, with a particular emphasis on the treatments that appear promising for use in future studies. Additionally, this review addresses HP bioreactor design, chondroprotective effects of HP, the use of HP for chondrogenic differentiation, the effects of high pressures, and HP mechanotransduction.

Extraction Techniques for the Decellularization of Tissue Engineered Articular Cartilage Constructs

Several prior studies have been performed to determine the feasibility of tissue decellularization to create a non-immunogenic xenogenic tissue replacement for bladder, vasculature, heart valves, knee meniscus, temporomandibular joint disc, ligament, and tendon. However, limited work has been performed with articular cartilage, and no studies have examined the decellularization of tissue engineered constructs. The objective of this study was to assess the effects of different decellularization treatments on articular cartilage constructs, engineered using a scaffoldless approach, after 4wks of culture, using a two-phased approach. In the first phase, five different treatments were examined: 1) 1% SDS, 2) 2% SDS, 3) 2% Tributyl Phosphate, 4) 2% Triton X-100, and 5) Hypotonic followed by hypertonic solution. These treatments were applied for either 1h or 8h, followed by a 2h wash in PBS. Following this wash, the constructs were assessed histologically, biochemically for cellularity, GAG, and collagen content, and biomechanically for compressive and tensile properties. In phase II, the best treatment from phase I was applied for 1, 2, 4, 6, or 8h in order to optimize the application time. Treatment with 2% SDS for 1h or 2h significantly reduced the DNA content of the tissue, while maintaining the biochemical and biomechanical properties. On the other hand, 2% SDS for 6h or 8h resulted in complete histological decellularization, with complete elimination of cell nuclei on histological staining, although GAG content and compressive properties were significantly decreased. Overall, 2% SDS, for 1 or 2h, appeared to be the most effective agent for cartilage decellularization, as it resulted in decellularization while maintaining the functional properties. The results of this study are exciting as they indicate the feasibility of creating engineered cartilage that may be non-immunogenic as a replacement tissue.

Synergistic and Additive Effects of Hydrostatic Pressure and Growth Factors on Tissue Formation

Background Hydrostatic pressure (HP) is a significant factor in the function of many tissues, including cartilage, knee meniscus, temporomandibular joint disc, intervertebral disc, bone, bladder, and vasculature. Though studies have been performed in assessing the role of HP in tissue biochemistry, to the best of our knowledge, no studies have demonstrated enhanced mechanical properties from HP application in any tissue. Methodology/Principal Findings The objective of this study was to determine the effects of hydrostatic pressure (HP), with and without growth factors, on the biomechanical and biochemical properties of engineered articular cartilage constructs, using a two-phased approach. In phase I, a 3×3 full-factorial design of HP magnitude (1, 5, 10 MPa) and frequency (0, 0.1, 1 Hz) was used, and the best two treatments were selected for use in phase II. Static HP at 5 MPa and 10 MPa resulted in significant 95% and 96% increases, respectively, in aggregate modulus (HA), with corresponding increases in GAG content. These regimens also resulted in significant 101% and 92% increases in Youngs modulus (EY), with corresponding increases in collagen content. Phase II employed a 3×3 full-factorial design of HP (no HP, 5 MPa static, 10 MPa static) and growth factor application (no GF, BMP-2+IGF-I, TGF-β1). The combination of 10 MPa static HP and TGF-β1 treatment had an additive effect on both HA and EY, as well as a synergistic effect on collagen content. This group demonstrated a 164% increase in HA, a 231% increase in EY, an 85% increase in GAG/wet weight (WW), and a 173% increase in collagen/WW, relative to control. Conclusions/Significance To our knowledge, this is the first study to demonstrate increases in the biomechanical properties of tissue from pure HP application, using a cartilage model. Furthermore, it is the only study to demonstrate additive or synergistic effects between HP and growth factors on tissue functional properties. These findings are exciting as coupling HP stimulation with growth factor application has allowed for the formation of tissue engineered constructs with biomechanical and biochemical properties spanning native tissue values.

Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs

OBJECTIVEnTo determine the effects of bone morphogenetic protein-2 (BMP-2), insulin-like growth factor (IGF-I), and transforming growth factor-beta1 (TGF-beta1) on the biochemical and biomechanical properties of engineered articular cartilage constructs under serum-free conditions.nnnMETHODSnA scaffoldless approach for tissue engineering, the self-assembly process, was employed. The study consisted of two phases. In the first phase, the effects of BMP-2, IGF-I, and TGF-beta1, at two concentrations and two dosage frequencies each were assessed on construct biochemical and biomechanical properties. In phase II, the effects of growth factor combination treatments were determined. Compressive and tensile mechanical properties, glycosaminoglycan (GAG) and collagen content, histology for GAG and collagen, and immunohistochemistry (IHC) for collagen types I and II were assessed.nnnRESULTSnIn phase I, BMP-2 and IGF-I treatment resulted in significant, >1-fold increases in aggregate modulus, accompanied by increases in GAG production. Additionally, TGF-beta1 treatment resulted in significant, approximately 1-fold increases in both aggregate modulus and tensile modulus, with corresponding increases in GAG and collagen content. In phase II, combined treatment with BMP-2 and IGF-I increased aggregate modulus and GAG content further than either growth factor alone, while TGF-beta1 treatment alone remained the only treatment to also enhance tensile properties and collagen content.nnnDISCUSSIONnThis study determined systematically the effects of multiple growth factor treatments under serum-free conditions, and is the first to demonstrate significant increases in both compressive and tensile biomechanical properties as a result of growth factor treatment. These findings are exciting as coupling growth factor application with the self-assembly process resulted in tissue engineered constructs with functional properties approaching native cartilage values.

Developing an Articular Cartilage Decellularization Process Toward Facet Joint Cartilage Replacement

OBJECTIVEThe facet joint has been identified as a significant source of morbidity in lower back pain. In general, treatments have focused on reducing the pain associated with facet joint osteoarthritis, and no treatments have targeted the development of a replacement tissue for arthritic facet articular cartilage. Therefore, the objective of this study was to develop a nonimmunogenic decellularized articular cartilage replacement tissue while maintaining functional properties similar to native facet cartilage tissue. METHODSIn vitro testing was performed on bovine articular cartilage explants. The effects of 2% sodium dodecyl sulfate (SDS), a detergent used for cell and nuclear membrane solubilization, on cartilage cellularity, biochemical, and biomechanical properties, were examined. Compressive biomechanical properties were determined using creep indentation, and the tensile biomechanical properties were obtained with uniaxial tensile testing. Biochemical assessment involved determination of the DNA content, glycosaminoglycan (GAG) content, and collagen content. Histological examination included hematoxylin and eosin staining for tissue cellularity, as well as staining for collagen and GAG. RESULTSTreatment with 2% SDS for 2 hours maintained the compressive and tensile biomechanical properties, as well as the GAG and collagen content while resulting in a decrease in cell nuclei and a 4% decrease in DNA content. Additionally, treatment for 8 hours resulted in complete histological decellularization and a 40% decrease in DNA content while maintaining collagen content and tensile properties. However, a significant decrease in compressive properties and GAG content was observed. Similar results were observed with 4 hours of treatment, although the decrease in DNA content was not as great as with 8 hours of treatment. CONCLUSIONTreatment with 2% SDS for 8 hours resulted in complete histological decellularization with decreased mechanical properties, whereas treatment for 2 hours maintained mechanical properties, but had a minimal effect on DNA content. Therefore, future studies must be performed to optimize a treatment for decellularization while maintaining mechanical properties close to those of facet joint cartilage. This study served as a step in creating a decellularized articular cartilage replacement tissue that could be used as a treatment for facet cartilage osteoarthritis.

Effects of Temporal Hydrostatic Pressure on Tissue-Engineered Bovine Articular Cartilage Constructs

The objective of this study was to determine the effects of temporal hydrostatic pressure (HP) on the properties of scaffoldless bovine articular cartilage constructs. The study was organized in three phases: First, a suitable control for HP application was identified. Second, 10 MPa static HP was applied at three different timepoints (6-10 days, 10-14 days, and 14-18 days) to identify a window in construct development when HP application would be most beneficial. Third, the temporal effects of 10-14-day static HP application, as determined in phase II, were assessed at 2, 4, and 8 weeks. Compressive and tensile mechanical properties, GAG and collagen content, histology for GAG and collagen, and immunohistochemistry for collagen types I and II were assessed. When a culture control identified in phase I was used in phase II, HP application from 10 to 14 days resulted in a significant 1.4-fold increase in aggregate modulus, accompanied by an increase in GAG content, while HP application at all timepoints enhanced tensile properties and collagen content. In phase III, HP had an immediate effect on GAG content, collagen content, and compressive stiffness, while there was a delayed increase in tensile stiffness. The enhanced tensile stiffness was still present at 8 weeks. For the first time, this study examined the immediate and long-term effects of HP on biomechanical properties, and demonstrated that HP has an optimal application time in construct development. These findings are exciting as HP stimulation allowed for the formation of robust tissue-engineered cartilage; for example, 10 MPa static HP resulted in an aggregate modulus of 273 +/- 123 kPa, a Youngs modulus of 1.6 +/- 0.4 MPa, a GAG/wet weight of 6.1 +/- 1.4%, and a collagen/wet weight of 10.6 +/- 2.4% at 4 weeks.

Biomechanical, biochemical, and histological characterization of canine lumbar facet joint cartilage: Laboratory investigation

OBJECTnTissue engineering appears to be a promising strategy for articular cartilage regeneration as a treatment for facet joint arthritis. Prior to the commencement of tissue engineering approaches, design criteria must be established to determine the required functional properties of the replacement tissue. As characterization of the functional properties of facet joint cartilage has not been performed previously, the objective of this study was to determine the biomechanical, biochemical, and histological properties of facet joint cartilage.nnnMETHODSnThe in vitro testing was conducted using 4 lumbar spinal segments obtained from skeletally mature canines. In each specimen, articular cartilage was obtained from the superior surface of the L3-4 and L4-5 facet joints. Creep indentation was used to determine the compressive biomechanical properties, while uniaxial tensile testing yielded the Young modulus and ultimate tensile strength of the tissue. Additionally, biochemical assessments included determinations of cellularity, glycosaminoglycan (GAG) content, and collagen content, as well as enzymelinked immunosorbent assays for collagen I and II production. Finally, histological characterization included H & E staining, as well as staining for collagen and GAG distributions.nnnRESULTSnThe means +/- standard deviation values were determined. There were no differences between the 2 spinal levels for any of the assessed properties. Averaged over both levels, the thickness was 0.49 +/- 0.10 mm and the hydration was 74.7 +/- 1.7%. Additionally, the cells/wet weight (WW) ratio was 6.26 +/- 2.66 x 10(4) cells/mg and the cells/dry weight (DW) ratio was 2.51 +/- 1.21 x 10(5) cells/mg. The GAG/WW was 0.038 +/- 0.013 and the GAG/ DW was 0.149 +/- 0.049 mg/mg, while the collagen/WW was 0.168 +/- 0.026 and collagen/DW was 0.681 +/- 0.154 mg/ mg. Finally, the aggregate modulus was 554 +/- 133 kPa, the Young modulus was 10.08 +/- 8.07 MPa, and the ultimate tensile strength was 4.44 +/- 2.40 MPa.nnnCONCLUSIONSnTo the best of the authors knowledge, this study is the first to provide a functional characterization of facet joint articular cartilage, thus providing design criteria for future tissue engineering studies.

Paradigms of Tissue Engineering with Applications to Cartilage Regeneration

Tissue engineering has been widely explored as an option for regeneration of various musculoskeletal tissues. This chapter examines the tissue engineering paradigm, or approach, with a focus on its application to cartilage tissue engineering. Since understanding the tissue engineering approach will require an understanding of cartilage physiology, a brief review of cartilage structure and function is provided. A discussion of current studies of the four parameters of the paradigm, namely scaffolds, cell sources, bioactive agents and bioreactors is presented, along with the latest technologies that incorporate manipulation of several parameters in a single approach.

Synergistic and Additive Effects of Hydrostatic Pressure and Growth Factor Application on Engineered Articular Cartilage Constructs

It has previously been demonstrated that hydrostatic pressure (HP) enhances the biochemical properties of self-assembled articular cartilage constructs [1]. However, studies that systematically assess the effects of HP magnitude and frequency are lacking. Additionally, studies examining the combined effects of hydrostatic pressure and growth factors are limited. To this end, this study sought to test the hypotheses that static HP will have the greatest enhancement of construct biomechanical and biochemical properties, and that there will be additive or synergistic effects when combining growth factors and HP stimulation.Copyright