R. Danilkowicz (Durham, US)

Duke University Medical Center

Presenter Of 6 Presentations

Podium Presentation Growth factors, PRP and Cytokines

10.2.6 - Histologic Grading Correlates With Inflammatory Biomarkers in Tibialis Posterior Tendon Dysfunction

Presentation Topic
Growth factors, PRP and Cytokines
Date
13.04.2022
Lecture Time
13:27 - 13:36
Room
Potsdam 3
Session Type
Free Papers
Disclosure
No Significant Commercial Relationship

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.screen shot 2021-10-14 at 11.20.26 am.pngscreen shot 2021-10-14 at 11.21.32 am.png

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.

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Podium Presentation Cartilage and Meniscus

10.2.8 - Histological and Inflammatory Cytokine Analysis of OLT After Failed Microfracture, a Comparison to Fresh Allograft Controls

Presentation Topic
Cartilage and Meniscus
Date
13.04.2022
Lecture Time
13:36 - 13:45
Room
Potsdam 3
Session Type
Free Papers
Disclosure
Dr. Adams: financial compensation from :stryker, conventus, exactech, DePuy, Medshape. Stock options from, Medshape, Restor3d. Dr. Easley: royalties, speaking compensation, consulting fees foe Exactech. Consulting fees and research support from Medartis

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.

new figure 4.jpgcombo.png

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.

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Podium Presentation Cartilage and Meniscus

18.3.4 - Synovial Fluid Fracture Hematoma Causes Cartilage Damage and Chondrocyte Death That is Partially Attenuated by Anti-Inflammatory Agents

Presentation Topic
Cartilage and Meniscus
Date
14.04.2022
Lecture Time
14:42 - 14:51
Room
Potsdam 1
Session Type
Free Papers
Disclosure
No Significant Commercial Relationship

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.

screen shot 2021-10-12 at 6.32.59 am.png

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.

screen shot 2021-10-12 at 6.32.44 am.png

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.

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Poster Cartilage Imaging and Functional Testing

P016 - MicroCT Analysis of Subchondral Bone Reveals Disorganized Architecture after Cartilage Damage

Presentation Topic
Cartilage Imaging and Functional Testing
Date
13.04.2022
Lecture Time
09:30 - 09:30
Room
Exhibition Foyer
Session Name
7.3 - Poster Viewing / Coffee Break / Exhibition
Session Type
Poster Session
Disclosure
No Significant Commercial Relationship

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.

figure 1 microct.png

figure 2 microct.png

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.

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Poster Biomaterials and Scaffolds

P041 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite Osteoblast Composite Hydrogels for Bone Tissue Engineering

Presentation Topic
Biomaterials and Scaffolds
Date
13.04.2022
Lecture Time
09:30 - 09:30
Room
Exhibition Foyer
Session Name
7.3 - Poster Viewing / Coffee Break / Exhibition
Session Type
Poster Session
Disclosure
No Significant Commercial Relationship

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).

ha figures.png

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.

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Poster Biomaterials and Scaffolds

P055 - 3D Bioprinted GelMA-Gelatin-Hydroxyapatite-Demineralized Bone Matrix Osteoblast Composite Hydrogels for Bone Tissue Engineering

Presentation Topic
Biomaterials and Scaffolds
Date
13.04.2022
Lecture Time
09:30 - 09:30
Room
Exhibition Foyer
Session Name
7.3 - Poster Viewing / Coffee Break / Exhibition
Session Type
Poster Session
Disclosure
No Significant Commercial Relationship

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.

screen shot 2021-10-11 at 10.54.57 am.png

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.

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Presenter Of 3 Presentations

Cartilage Imaging and Functional Testing

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.

figure 1 microct.png

figure 2 microct.png

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.

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Biomaterials and Scaffolds

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).

ha figures.png

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.

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Biomaterials and Scaffolds

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.

screen shot 2021-10-11 at 10.54.57 am.png

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.

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