R. Danilkowicz (Durham, US)
Duke University Medical CenterPresenter Of 6 Presentations
10.2.6 - Histologic Grading Correlates With Inflammatory Biomarkers in Tibialis Posterior Tendon Dysfunction
Abstract
Purpose
The pathophysiology of pain in Tibialis posterior tendon dysfunction (TPTD) is not well understood. It has been theorized that TPTD is a degenerative process unrelated to inflammation. However, we hypothesize that inflammation is a key component of TPTD. The purpose of this study was to determine if inflammatory cytokines, matrix metalloproteases (MMPs), and glutamate were elevated in diseased TPTs.
Methods and Materials
Matched torn TPT, TPT insertion, and flexor digitorum longus (FDL) samples were collected from 21 patients with TPTD. The samples were individually incubated in media for 48 hours. The conditioned media was analyzed for inflammatory cytokines, MMPs, and glutamate. Histology was performed on the samples. Statistical analysis was performed with Friedman’s test and Wilcoxon-signed-rank post-hoc tests with Bonferroni correction (α = 0.0167). Spearman’s ρ was used to determine non-parametric correlations between histology and cytokine, MMP, and glutamate concentrations.
Results
Diseased TPT and TPT insertion groups were significantly elevated compared to healthy FDL for inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α and MMPs MMP-1, MMP-2, and MMP-3 (p<0.005). Differences in glutamate concentrations were also significant, but only the diseased TPT group was significantly elevated compared to the healthy FDL tendons (p<0.01). Histologic grading correlated with inflammatory cytokine levels.
Conclusion
Diseased TPT and the TPT insertion demonstrated significantly elevated levels of inflammatory cytokines and MMPs compared to healthy FDL controls, suggesting a role for inflammation in the disease process. The amount of inflammation correlated with increased tendon degradation. The TPT, but not the insertion, contained significantly larger amounts of glutamate.
10.2.8 - Histological and Inflammatory Cytokine Analysis of OLT After Failed Microfracture, a Comparison to Fresh Allograft Controls
Abstract
Purpose
The purpose of this study is to characterize the structural and biochemical makeup of failed microfracture lesions in an effort to identify potential reasons for continued pain and poor OLT healing after microfracture as well as identify potential avenues for future research and early intervention.
Methods and Materials
Eight specimens were analyzed from symptomatic OLTs after microfracture who later underwent fresh osteochondral allograft transplantation. For each patient, the failed microfracture specimen and a portion of the fresh allograft replacement tissue were collected. The allograft served as a control. Histology of the failed microfracture and the allograft replacement were scored using the Osteoarthritis Research Society International (OARSI) system. Surface roughness was also compared. In addition, tissue culture supernatants were analyzed for sixteen secreted cytokines and matrix metalloproteinases (MMPs) responsible for inflammation, pain, cartilage damage, and chondrocyte death.
Results
The OARSI grade, stage, and total score as well as surface smoothness were significantly lower in the failed microfracture sample, indicating better cartilage and bone morphology for the allografts compared to the failed microfracture lesions. Analyzed cytokines and MMPs were significantly elevated in the microfracture tissue culture supernatants when compared to fresh osteochondral tissue supernatants.
Conclusion
The current study is the first to provide histologic and secretomic evidence demonstrating insufficiencies of osteochondral tissue after failed microfracture. These data demonstrate a significantly rougher cartilage surface, cartilage and subchondral bone histology that more closely resembles osteoarthritis, and elevated inflammatory cytokines and matrix metalloproteinases responsible for pain, inflammation, cartilage damage, and chondrocyte death when compared to fresh osteochondral allografts used as controls.
18.3.4 - Synovial Fluid Fracture Hematoma Causes Cartilage Damage and Chondrocyte Death That is Partially Attenuated by Anti-Inflammatory Agents
Abstract
Purpose
Intra-articular ankle fracture (IAF) causes post-traumatic osteoarthritis (PTOA) but the exact mechanism is unknown. Pro-inflammatory mediators have been shown to be present in the synovial fluid fracture hematoma (SFFH), but have not been linked to cartilage damage. The purpose of this study was to determine if the SFFH causes cartilage damage and whether this damage can be attenuated by commercially available therapeutic agents.
Methods and Materials
SF was obtained from 54 intra-articular ankle fractures and cultured with cartilage discs from the dome of fresh allograft human tali and randomly assigned to one of the following groups: (A) control—media only, (B) SFFH from days 0-2 after fracture, (C) SFFH from days 3-9, and (D) SFFH from days 10-14, (E) group B + IL-1Ra, and (F) group B + doxycycline. The cartilage discs underwent histological evaluation for cell survival and cartilage matrix components. The spent media were analyzed for inflammatory mediators.
Results
Cartilage discs cultured with SFFH in groups B, C, and D demonstrated significantly increased production of cytokines, metalloptoteinases (MMPs), and extracellular matrix breakdown products. Safranin O staining was significantly decreased in group B. The negative effects on cartilage were partially attenuated with the addition of either IL-1RA or doxycycline. There was no difference in chondrocyte survival among the groups.
Conclusion
Exposure of uninjured cartilage to IAF SFFH caused activation of cartilage damage pathways evident through cartilage disc secretion of inflammatory cytokines, MMPs, and cartilage matrix fragments. The addition of IL-1Ra or doxycycline to SFFH culture partially attenuated this response.
P016 - MicroCT Analysis of Subchondral Bone Reveals Disorganized Architecture after Cartilage Damage
Abstract
Purpose
There are numerous treatment modalities of the ankle and knee that work to address cartilage damage. The purpose of our study is to determine the extent of damage in the subchondral bone after chondral injuries as it relates to the bony architecture to provide evidence for why certain treatment modalities may fail.
Methods and Materials
Samples were fixed in 10% formalin for 48 h then transferred to 70% ethanol. Quantitative three-dimensional evaluation of the samples was undertaken by μCT using a Viva CT 80 (Scanco, Brüttisellen, Switzerland) at 55 kVp and 145 μA with a resolution of 15.6 μm voxel size. A hydroxyapatite calibration phantom was used to scale values of linear attenuation for the calcified tissues to bone density values (mg/cm3). Calcified tissues were segmented using a thresholding procedure in which bone was defined by a threshold above 500mg HA/cm3.
Results
Of the four diseased samples, the average relative bone volume (%) was 11.86. Comparatively, the four allograft controls contained a relative bone volume (%) of 16.54. Two diseased samples did not have allograft controls for direct comparison.
Conclusion
Damaged subchondral bone is a known source of continued pain, declining results, requirement for additional procedures, and OA progression after injury. This study is the first of its kind to utilize microCT to analyze damaged osteochondral samples in an attempt to quantify the damage.
P041 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite Osteoblast Composite Hydrogels for Bone Tissue Engineering
Abstract
Purpose
The purpose of this study was to investigate the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), and osteoblasts for bone tissue engineering.
Methods and Materials
A mouse calvarial osteoblast-laden GelMA-gelatin bioink consisting of various concentrations of HA was 3D-bioprinted into porous hydrogel constructs. The constructs were cross-linked via photopolymerization and cultured in osteogenic medium. After 1, 14, and 28 days, the constructs were analyzed. The water weight percent differences of the hydrogels were characterized. An ALP assay and histological analysis were performed. Cell survivability and proliferation in the composite hydrogels was determined. Real-time polymerase chain reaction was performed to measure expression levels of osteogenic genes, BMP-7, and osteocalcin relative to a housekeeping gene (GAPDH).
Results
The addition of 5, 10, and 20 mg/ml of HA reduced hydrogel swelling from baseline GelMA-Gelatin hydrogels (p ≤ 0.01). HA decreased hydrogel breakdown in a concentration dependent manner (p ≤ 0.001). Alamar Blue assay demonstrated significantly increased cell proliferation. There was no difference in metabolic activity among the groups (p ≤ 0.01). The addition of 5mg/ml and 20mg/ml of HA significantly increased ALP expression at 7 and 28 days (p ≤ 0.05). Live/dead staining showed the majority of osteoblasts survived in all groups at 1, 14, and 28 days. The addition of 20mg/ml of HA (GG20HA) demonstrated greater BMP7 and BGLAP gene expression at both 14 and 28 days over hydrogels without HA (p ≤ 0.05).
Conclusion
The addition of HA to GelMA-gelatin hydrogels significantly decreased hydrogel swelling, improved the ability to resist enzymatic degradation, increased osteoblastic differentiation and mineralization, and increased osteogenic gene expression while maintaining equal cell viability and proliferation to non-HA hydrogels. These findings support a lower threshold of 20 mg/ml of HA as an optimal concentration to support gene expression associated with osteoblast cell differentiation and maturation.
P055 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite-Demineralized Bone Matrix Osteoblast Composite Hydrogels for Bone Tissue Engineering
Abstract
Purpose
Tissue engineering via 3D bioprinting offers a novel solution to treating large bone voids. The optimum bioink for bone tissue engineering is unclear. The purpose of this study was to investigate the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), demineralized bone matrix (DBM) and osteoblasts for bone tissue engineering.
Methods and Materials
A mouse calvarial osteoblast-laden GelMA-gelatin-HA bioink consisting of various concentrations of DBM was 3D-bioprinted into porous hydrogel constructs. The 3D-fabricated constructs were cross-linked via photopolymerization and cultured in osteogenic medium. After 1, 14, and 28 days, a cohort of constructs were analyzed. The water weight percent differences of the hydrogels were characterized following fabrication along with the degradation behavior in standard culture medium for 28 days. Cell survivability and proliferation was determined.
Results
The addition of DBM to the bioink significantly decreased water content (%) from baseline GelMA-Gelatin-HA hydrogels with a significant trend of decreased swelling with increasing DBM content. The addition of 40, 80, and 120 mg/ml of DBM significantly reduced hydrogel swelling ratios (p ≤ 0.0001). DBM decreased hydrogel breakdown in a concentration dependent manner. Alamar Blue assay demonstrated significantly increased cell proliferation across groups. There was a slight trend of increasing metabolic activity with increasing DBM content. Live/dead staining at 1, 14, and 28 days after printing showed the majority of the osteoblasts were alive at each time-point.
Conclusion
Addition of DBM significantly decreased hydrogel swelling and suggests DBM to be essential in construct stability. DBM may also be altered to modulate the rate of degradation in vitro. Additionally, Alamar Blue results indicate that DBM addition to GelMA-Gelatin-HA bioink did not adversely affect cell viability and cells were able to survive the bioprinting process. These findings support GelMA-gelatin-HA-DBM as a viable bioink for bone tissue engineering.
Presenter Of 3 Presentations
P016 - MicroCT Analysis of Subchondral Bone Reveals Disorganized Architecture after Cartilage Damage
Abstract
Purpose
There are numerous treatment modalities of the ankle and knee that work to address cartilage damage. The purpose of our study is to determine the extent of damage in the subchondral bone after chondral injuries as it relates to the bony architecture to provide evidence for why certain treatment modalities may fail.
Methods and Materials
Samples were fixed in 10% formalin for 48 h then transferred to 70% ethanol. Quantitative three-dimensional evaluation of the samples was undertaken by μCT using a Viva CT 80 (Scanco, Brüttisellen, Switzerland) at 55 kVp and 145 μA with a resolution of 15.6 μm voxel size. A hydroxyapatite calibration phantom was used to scale values of linear attenuation for the calcified tissues to bone density values (mg/cm3). Calcified tissues were segmented using a thresholding procedure in which bone was defined by a threshold above 500mg HA/cm3.
Results
Of the four diseased samples, the average relative bone volume (%) was 11.86. Comparatively, the four allograft controls contained a relative bone volume (%) of 16.54. Two diseased samples did not have allograft controls for direct comparison.
Conclusion
Damaged subchondral bone is a known source of continued pain, declining results, requirement for additional procedures, and OA progression after injury. This study is the first of its kind to utilize microCT to analyze damaged osteochondral samples in an attempt to quantify the damage.
P041 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite Osteoblast Composite Hydrogels for Bone Tissue Engineering
Abstract
Purpose
The purpose of this study was to investigate the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), and osteoblasts for bone tissue engineering.
Methods and Materials
A mouse calvarial osteoblast-laden GelMA-gelatin bioink consisting of various concentrations of HA was 3D-bioprinted into porous hydrogel constructs. The constructs were cross-linked via photopolymerization and cultured in osteogenic medium. After 1, 14, and 28 days, the constructs were analyzed. The water weight percent differences of the hydrogels were characterized. An ALP assay and histological analysis were performed. Cell survivability and proliferation in the composite hydrogels was determined. Real-time polymerase chain reaction was performed to measure expression levels of osteogenic genes, BMP-7, and osteocalcin relative to a housekeeping gene (GAPDH).
Results
The addition of 5, 10, and 20 mg/ml of HA reduced hydrogel swelling from baseline GelMA-Gelatin hydrogels (p ≤ 0.01). HA decreased hydrogel breakdown in a concentration dependent manner (p ≤ 0.001). Alamar Blue assay demonstrated significantly increased cell proliferation. There was no difference in metabolic activity among the groups (p ≤ 0.01). The addition of 5mg/ml and 20mg/ml of HA significantly increased ALP expression at 7 and 28 days (p ≤ 0.05). Live/dead staining showed the majority of osteoblasts survived in all groups at 1, 14, and 28 days. The addition of 20mg/ml of HA (GG20HA) demonstrated greater BMP7 and BGLAP gene expression at both 14 and 28 days over hydrogels without HA (p ≤ 0.05).
Conclusion
The addition of HA to GelMA-gelatin hydrogels significantly decreased hydrogel swelling, improved the ability to resist enzymatic degradation, increased osteoblastic differentiation and mineralization, and increased osteogenic gene expression while maintaining equal cell viability and proliferation to non-HA hydrogels. These findings support a lower threshold of 20 mg/ml of HA as an optimal concentration to support gene expression associated with osteoblast cell differentiation and maturation.
P055 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite-Demineralized Bone Matrix Osteoblast Composite Hydrogels for Bone Tissue Engineering
Abstract
Purpose
Tissue engineering via 3D bioprinting offers a novel solution to treating large bone voids. The optimum bioink for bone tissue engineering is unclear. The purpose of this study was to investigate the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), demineralized bone matrix (DBM) and osteoblasts for bone tissue engineering.
Methods and Materials
A mouse calvarial osteoblast-laden GelMA-gelatin-HA bioink consisting of various concentrations of DBM was 3D-bioprinted into porous hydrogel constructs. The 3D-fabricated constructs were cross-linked via photopolymerization and cultured in osteogenic medium. After 1, 14, and 28 days, a cohort of constructs were analyzed. The water weight percent differences of the hydrogels were characterized following fabrication along with the degradation behavior in standard culture medium for 28 days. Cell survivability and proliferation was determined.
Results
The addition of DBM to the bioink significantly decreased water content (%) from baseline GelMA-Gelatin-HA hydrogels with a significant trend of decreased swelling with increasing DBM content. The addition of 40, 80, and 120 mg/ml of DBM significantly reduced hydrogel swelling ratios (p ≤ 0.0001). DBM decreased hydrogel breakdown in a concentration dependent manner. Alamar Blue assay demonstrated significantly increased cell proliferation across groups. There was a slight trend of increasing metabolic activity with increasing DBM content. Live/dead staining at 1, 14, and 28 days after printing showed the majority of the osteoblasts were alive at each time-point.
Conclusion
Addition of DBM significantly decreased hydrogel swelling and suggests DBM to be essential in construct stability. DBM may also be altered to modulate the rate of degradation in vitro. Additionally, Alamar Blue results indicate that DBM addition to GelMA-Gelatin-HA bioink did not adversely affect cell viability and cells were able to survive the bioprinting process. These findings support GelMA-gelatin-HA-DBM as a viable bioink for bone tissue engineering.