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

PicoGreen results depicting changes in double stranded (ds)DNA amounts in articular cartilage throughout the decellularization process.

<p>Processing the cartilage with both physical and chemical methods significantly reduced the amount of dsDNA in the matrix by 86%. * denotes significance (p<0.01) from native cartilage (n = 6). All results are reported as mean ± standard deviation.</p

SEM Image of Cryo-ground DCC.

<p>The scale bar is 1 mm.</p

Hydroxyproline content of cartilage matrix during physical and chemical decellularization process.

<p>No statically significant differences were observed during the processing (n = 6). All results are reported as mean ± standard deviation.</p

Relative expression of chondrogenic and osteogenic gene markers (n = 5).

<p>A) collagen II, B) Sox-9, C) aggrecan, D) collagen X, E) Runx-2, and F) collagen I. @ denotes significant difference from control group at same time point, # denotes significant difference from TGF-β group at same time point, * denotes significant difference between day 1 value, + denotes significant difference from previous time point, $ denotes significant difference from DCC group at same time point.</p

DNA content of cell pellets at days 1 and 7 (n = 5).

<p>All groups significantly increased DNA content between 1 and 7 days. * p<0.05 between day 1 and 7, ** p<0.01 between day 1 and 7, A = p<0.05 between DCC and control, and B = p<0.01 between DCC and DVC. All results are reported as mean ± standard deviation.</p