Immobilization using external or internal splints is a standard and effective procedure to treat minor skeletal fractures. In the case of major skeletal defects caused by extreme trauma, infectious diseases or tumors, the surgical implantation of a bone graft from external sources is required for a complete cure. Practical disadvantages, such as the risk of immune rejection and infection at the implant site, are high in xenografts and allografts. Currently, an autograft from the iliac crest of a patient is considered the “gold standard” method for treating large-scale skeletal defects. However, this method is not an ideal solution due to its limited availability and significant reports of morbidity in the harvest site (30%) as well as the implanted site (5–35%). Tissue-engineered bone grafts aim to create a mechanically strong, biologically viable and degradable bone graft by combining a three-dimensional porous scaffold with osteoblast or progenitor cells. The materials used for such tissue-engineered bone grafts can be broadly divided into ceramic materials (calcium phosphates) and biocompatible/bioactive synthetic polymers. This review summarizes the types of materials used to make scaffolds for cryo-preservable tissue-engineered bone grafts as well as the distinct methods adopted to create the scaffolds, including traditional scaffold fabrication methods (solvent-casting, gas-foaming, electrospinning, thermally induced phase separation) and more recent fabrication methods (fused deposition molding, stereolithography, selective laser sintering, Inkjet 3D printing, laser-assisted bioprinting and 3D bioprinting). This is followed by a short summation of the current osteochondrogenic models along with the required scaffold mechanical properties for in vivo applications. We then present a few results of the effects of freezing and thawing on the structural and mechanical integrity of PLLA scaffolds prepared by the thermally induced phase separation method and conclude this review article by summarizing the current regulatory requirements for tissue-engineered products.

Bio-degradable Poly (l-lactic acid) (PLLA) scaffolds were prepared by using thermal liquid-liquid phase separation method. A solution of PLLA-Dioxane was prepared by dissolving PLLA in dehydrated 1,4-Dioxane. This PLLA-Dioxane solution was then directionally frozen in vials of similar dimensions but made of different materials (borosilicate glass, aluminum and copper). The frozen solution was then placed in a freeze-dryer to allow for the frozen dioxane to sublimate or lyophilized. The porosities of the resulting PLLA scaffolds were calculated and their porous structures were studied and compared between the three different vials (and the corresponding cooling rates).

Bio-degradable poly (l-lactic acid) (PLLA) scaffolds were prepared by using thermally induced phase separation (TIPS) method. A solution of PLLA-Dioxane was formed by dissolving PLLA in dehydrated 1,4-Dioxane at three wt/vol percentages, specifically 3, 7 and 10%. This PLLA-Dioxane solution was then frozen in borosilicate glass vials (5mL) at three cooling rates (1, 10 and 40 °C/min) in a commercially available controlled rate freezer (CRF). The frozen solution was freeze-dried to sublimate the dioxane. The microstructural properties of the resulting PLLA scaffolds were determined utilizing Scanning Electron Microscopy (SEM) images and uniaxial compressive testing. The relationship between the wt/vol ratio of PLLA and Dioxane and the imposed cooling rates on the structural properties of PLLA scaffolds was determined.

Poly (l-lactic acid) (PLLA) scaffolds for bone grafts were prepared by using Thermally Induced Phase Separation (TIPS) method. Solutions of PLLA-dioxane was formed by dissolving a pre-determined value of PLLA in dioxane (3, 7 and 10% wt/vol) at 323K. These solutions were frozen at controlled cooling rates (1, 10 and 40°C/min) in cylindrical capsules. The frozen solutions were freeze-dried for a period of 48 hours for the frozen dioxane to completely sublimate, leaving the porous PLLA scaffold. The scaffolds which were formed under different processing conditions were characterized in terms of porosity, pore sizes and compressive moduli.

Bio-degradable Poly (l-lactic acid) (PLLA) scaffolds synthesized using thermally induced phase separation (TIPS) method was used to load cryo-preserved human adipose derived stem cells (hASCs). To make the scaffolds, PLLA-Dioxane solutions were formed by dissolving PLLA in 1,4-Dioxane with three different compositions (wt/vol). These PLLA-Dioxane solutions, frozen in three different cooling rates were lyophilized at 0.037bar and −70°C for 48hrs resulting in porous PLLA scaffolds. Based on the porosity, pore size and compressive strength, a suitable scaffold was chosen to investigate its bio-compatibility and osteo-inductive potential.