Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

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Colloidal gels encapsulating natural materials and exhibiting paste-like properties for placement are promising for filling complex geometries in craniofacial bone regeneration applications. Colloidal materials have demonstrated modest clinical outcomes as bone substitutes in orthopedic applications, but limited success in craniofacial applications. As such, development of a novel colloidal gel will fill a void in commercially available products for use in craniofacial reconstruction. One likely application for this technology is cranial reconstruction. Currently, traumatic brain injury (TBI) is often treated with a hemi-craniectomy, a procedure in which half the cranium is removed to allow the injured brain to swell and herniate beyond the enclosed cranial vault. The use of colloidal gels would allow for the design of a pliable material capable of expansion during brain swelling and facilitate cranial bone regeneration alleviating the need for a second surgery to replace the previously removed hemi-cranium. In the current study, colloidal nanoparticles of hydroxyapatite (HAp), demineralized bone matrix (DBM), and decellularized cartilage (DCC) were combined with hyaluronic acid (HA) to form colloidal gels with desirable rheological properties ([Formula: see text] ≥ 100 Pa). BMP-2 and VEGF growth factors were included to assess extracellular matrix (ECM) contribution of DBM and DCC. The HA-HAp (BMP-2) and HA-HAp-DCC group had 89 and 82% higher bone regeneration compared to the sham group, respectively (p < 0.01). Material retention issues observed may be alleviated by implementing chemical crosslinking. Overall, DCC may be a promising material for bone regeneration in general, and colloidal gels may hold significant potential in craniofacial applications.

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) (Mw = 15–30 kDa) and hyaluronic acid (HA) (Mw = 20–41 kDa) can be used to modify several rheological properties including consistency index (K), flow-behavior index (n), and yield stress (τy) of submicrometer hydroxyapatite (HAP) (Davg ≤ 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 (KGAG-HAP ≥ KGAG + KHAP) without compromising shear-thinning behavior (n < 1) of the gel. In addition, GAG–HAP colloids containing high concentrations of HAP (60–80% w/v) exhibited substantial yield stress (τy ≥ 100 Pa) and viscoelastic recovery properties (G′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.

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) microparticles, i.e., demineralized bone matrix (DBM) and decellularized cartilage (DCC), with hyaluronic acid (HA) and hydroxyapatite (HAP) nanoparticles, 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 (G' ≈ 100-10 000 Pa), yield stresses (τ ≈ 100-1000 Pa), and viscoelastic recoveries (G' ≥ 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 G' and a mean 4-fold increase in τ 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.