Mapping Glycosaminoglycan–Hydroxyapatite Colloidal Gels as Potential Tissue Defect Fillers

Malleable biomaterials such as Herschel–Bulkley (H–B) fluids possess shear responsive rheological properties and are capable of self-assembly and viscoelastic recovery following mechanical disruption (e.g., surgical placement via injection or spreading). This study demonstrated that the addition of moderate molecular weight glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) (<i>M</i>_w = 15–30 kDa) and hyaluronic acid (HA) (<i>M</i>_w = 20–41 kDa) can be used to modify several rheological properties including consistency index (<i>K</i>), flow-behavior index (<i>n</i>), and yield stress (τ_y) of submicrometer hydroxyapatite (HAP) (<i>D</i>_avg ≤ 200 nm) colloidal gels. GAG–HAP colloidal mixtures exhibited substantial polymer–particle synergism, likely due to “bridging” flocculation, which led to a synergistic increase in consistency index (<i>K</i>_GAG‑HAP ≥ <i>K</i>_GAG + <i>K</i>_HAP) without compromising shear-thinning behavior (<i>n</i> < 1) of the gel. In addition, GAG–HAP colloids containing high concentrations of HAP (60–80% w/v) exhibited substantial yield stress (τ_y ≥ 100 Pa) and viscoelastic recovery properties (<i>G</i>′_recovery ≥ 64%). While rheological differences were observed between CS–HAP and HA–HAP colloidal gels, both CS and HA represent feasible options for future studies involving bone defect filling. Overall, this study identified mixture regions where rheological properties in CS–HAP and HA–HAP colloidal gels aligned with desired properties to facilitate surgical placement in non-load-bearing tissue-filling applications such as calvarial defects

Hyaluronic-Acid–Hydroxyapatite Colloidal Gels Combined with Micronized Native ECM as Potential Bone Defect Fillers

One of the grand challenges in translational regenerative medicine is the surgical placement of biomaterials. For bone regeneration in particular, malleable and injectable colloidal gelsare frequently designed to exhibit self-assembling and shear-response behavior which facilitates biomaterial placement in tissue defects. The current study demonstrated that by combining native extracellular matrix (ECM) <i>microparticles</i>, i.e., demineralized bone matrix (DBM) and decellularized cartilage (DCC), with hyaluronic acid (HA) and hydroxyapatite (HAP) <i>nanoparticles</i>, a viscoelastic colloidal gel consisting exclusively of natural materials was achieved. Rheological testing of HA-ECM suspensions and HA-HAP-ECM colloidal gels concluded either equivalent or substantially higher storage moduli (<i>G</i>′ ≈ 100–10 000 Pa), yield stresses (τ_<i>y</i> ≈ 100–1000 Pa), and viscoelastic recoveries (<i>G</i>′_recovery ≥ 87%) in comparison with controls formulated without ECM, which indicated a previously unexplored synergy in fluid properties between ECM microparticles and HA-HAP colloidal networks. Notable rheological differences were observed between respective DBM and DCC formulations, specifically in HA-HAP-DBM mixtures, which displayed a mean 3-fold increase in <i>G</i>′ and a mean 4-fold increase in τ_<i>y</i> from corresponding DCC mixtures. An initial in vitro assessment of these potential tissue fillers as substrates for cell growth revealed that all formulations of HA-ECM and HA-HAP-ECM showed no signs of cytotoxicity and appeared to promote cell viability. Both DBM and DCC colloidal gels represent promising platforms for future studies in bone and cartilage tissue engineering. Overall, the current study identified colloidal gels constructed exclusively of natural materials, with viscoelastic properties that may facilitate surgical placement for a wide variety of therapeutic applications

Representative images for immunohistochemistry analysis for collagen I, collagen II, and aggrecan staining for hWJC groups at week 0, 2, and 3.

<p>The 10M LA and 20M HA groups at week 0 displayed the highest staining intensity for aggrecan and 20M LA group at week 3 displayed the highest staining intensity for aggrecan. Scale bar = 200 μm.</p

DNA content for all the rBMSC and hWJC groups at 0, 2, and 3 weeks expressed as DNA (μg/ scaffold).

<p>(A) DNA content of all the rBMSC LA and LA CS groups. (B) DNA content of all the rBMSC HA and HA CS groups. (C) DNA content of all the hWJC LA and LA CS groups. (D) DNA content of all the hWJC HA and HA CS groups. All aggregate groups had statistically significant increase in DNA over week 0, HA groups had significantly higher values compared to the CS control groups at week 3. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference from the week 0 value. (#) represents statistically significant difference from the previous time point and (**) represents statistically significant difference from the control at that time point (p < 0.05).</p

GAG content for all the rBMSC and hWJC groups at 0, 2, and 3 weeks expressed as GAG/DNA (A&B).

<p>All rBMSC aggregate groups had statistically significant increase in GAG/DNA over week 0, and select aggregates groups had significantly higher values compared to the control groups at week 3. GAG content for all the hWJC groups at week expressed as GAG/ DNA (C&D) at week 0, 2, and 3. 10 M HA at week 2 exhibited the highest GAG/DNA value and decreased at week 3. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference from the week 0 value. (#) represents statistically significant difference from the previous time point and (**) represents statistically significant difference from the control at that time point. (p < 0.05).</p

Representative images for immunohistochemistry analysis for collagen I, collagen II, and aggrecan staining for rBMSC groups at weeks 0, 2, and 3.

<p>At week 2, 20M LA had the most intense staining for collagen II and aggrecan. At week 3, 10M LA had the highest staining intensity at collagen II. Scale bar = 200 μm.</p

Explanation of the different experimental groups used in the study and their abbreviations.

<p>Explanation of the different experimental groups used in the study and their abbreviations.</p

Collagen content for all the rBMSC and hWJC groups expressed as collagen/DNA (A&B) at week 0, 2, and 3.

<p>All rBMSC aggregate groups had statistically significant increase in collagen over week 0. At week 3, 20 M HA also displayed the highest collagen/DNA. Collagen content for all the hWJC groups expressed as collagen/DNA (C&D) at week 0, 2, and 3. We noticed that the 20 M HA at week 0 had the highest collagen/DNA value. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference over the week 0 value. (#) represents statistically significant highest value of the particular group and (**) represents statistically significant difference from the control at that time point. (p < 0.05).</p

Cell viability at weeks 0 and 3.

<p>(A) Live-dead images of the 10M/mL hWJC aggregates and controls. (B) Live dead images of the 20M/mL hWJC aggregates. Scale bar = 100 μm.</p

Summary of the best performing groups for all the functional assays.

<p>Summary of the best performing groups for all the functional assays.</p