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Abstract: Macropores in tissue engineering scaffolds provide space for vascularization, cell-proliferation and cellular interactions, and is crucial for successful tissue regeneration. Modulating the size and density of macropores may promote desirable cellular processes at different stages of tissue development. Most current techniques for fabricating macroporous scaffolds produce fixed macroporosity and do not allow the control of porosity during cell culture. Most macropore-forming techniques also involve non-physiological conditions, such that cells can only be seeded in a post-fabrication process, which often leads to low cell seeding efficiency and uneven cell distribution. Here we report a process to create dynamic hydrogels as tissue engineering scaffolds with tunable macroporosity using stimuli-responsive porogens of gelatin, alginate and hyaluronic acid, which degrade in response to specific stimuli including temperature, chelating and enzymatic digestion, respectively. SEM imaging confirmed sequential pore formation in response to sequential stimulations: 37 °C on day 0, EDTA on day 7, and hyaluronidase on day 14. Bovine chondrocytes were encapsulated in the Alg porogen, which served as cell-delivery vehicles, and changes in cell viability, proliferation and tissue formation during sequential stimuli treatments were evaluated. Our results showed effective cell release from Alg porogen with high cell viability and markedly increased cell proliferation and spreading throughout the 3D hydrogels. Dynamic pore formation also led to significantly enhanced type II and X collagen production by chondrocytes. This platform provides a valuable tool to create stimuli-responsive scaffolds with dynamic macroporosity for a broad range of tissue engineering applications, and may also be used for fundamental studies to examine cell responses to dynamic niche properties. [Copyright &y& Elsevier]

Abstract: Calcium phosphate cement (CPC) is highly promising for clinical uses due to its in situ-setting ability, excellent osteoconductivity and bone-replacement capability. However, the low strength limits its use to non-load-bearing applications. The objectives of this study were to develop a layered CPC structure by combining a macroporous CPC layer with a strong CPC layer, and to investigate the effects of porosity and layer thickness ratios. The rationale was for the macroporous layer to accept tissue ingrowth, while the fiber-reinforced strong layer would provide the needed early-strength. A biopolymer chitosan was incorporated to strengthen both layers. Flexural strength, S (mean±sd; n=6) of CPC-scaffold decreased from (9.7±1.2) to (1.8±0.3)MPa (p<0.05), when the porosity increased from 44.6% to 66.2%. However, with a strong-layer reinforcement, S increased to (25.2±6.7) and (10.0±1.4)MPa, respectively, at these two porosities. These strengths matched/exceeded the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. Relationships were established between S and the ratio of strong layer thickness/specimen thickness, a/h:S=(17.6 a/h+3.2)MPa. The scaffold contained macropores with a macropore length (mean±sd; n=147) of (183±73)μm, suitable for cell infiltration and tissue ingrowth. Nano-sized hydroxyapatite crystals were observed to form the scaffold matrix of CPC with chitosan. In summary, a layered CPC implant, combining a macroporous CPC with a strong CPC, was developed. Mechanical strength and macroporosity are conflicting requirements. However, the novel functionally graded CPC enabled a relatively high strength and macroporosity to be simultaneously achieved. Such an in situ-hardening nano-apatite may be useful in moderate stress-bearing applications, with macroporosity to enhance tissue ingrowth and implant resorption. [Copyright &y& Elsevier]

Calcium phosphate cement (CPC) sets to form hydroxyapatite and has been used in medical and dental procedures. However, the brittleness and low strength of CPC prohibit its use in many stress-bearing locations, unsupported defects, or reconstruction of thin bones. Recent studies incorporated fibers into CPC to improve its strength. In the present study, a novel methodology was used to combine the reinforcement with macroporosity: large-diameter resorbable fibers were incorporated into CPC to provide short-term strength, then dissolved to create macropores suitable for bone ingrowth. Two types of resorbable fibers with 322 μm diameters were mixed with CPC to a fiber volume fraction of 25%. The set specimens were immersed in saline at 37°C for 1, 7, 14, 28 and 56 d, and were then tested in three-point flexure. SEM was used to examine crack-fiber interactions. CPC composite achieved a flexural strength 3 times, and work-of-fracture (toughness) nearly 100 times, greater than unreinforced CPC. The strength and toughness were maintained for 2–4 weeks of immersion, depending on fiber dissolution rate. Macropores or channels were observed in CPC composite after fiber dissolution. In conclusion, incorporating large-diameter resorbable fibers can achieve the needed short-term strength and fracture resistance for CPC while tissue regeneration is occurring, then create macropores suitable for vascular ingrowth when the fibers are dissolved. The reinforcement mechanisms appeared to be crack bridging and fiber pullout; the mechanical properties of the CPC matrix also affected the composite properties. [Copyright &y& Elsevier]