PoLyMER SCAFFOLD PROCESSING Robert C. Thomson, Albert K. Shung, Michael J. Yaszemski, and Antonios G. Mikos* INTRODUCTION Jolymer scaffolds must possess many key characteristics, including high porosity and surface area, structural strength, and specific three-dimensional shapes, ro be useful as materials fr tissue ‘engineering. These characteristics are determined by the scaffold fabrication technique, which must be developed such that it does not adversely affect the biocompatibility ofthe material of construction. Novel processing techniques have been developed to manufacture scaffolds with desirable and reproducible characteristics, which have been used to demonstrate the feasibility of regenerating human tissue. These polymer scaffold fabrication techniques have been tailored to create scafolds with particular characteristics that will satisfy the critical tissue engineering re- quirements of specific organs. Restoration of organ function by utilizing tissue engineering technologies often requires the use ofa temporary porous scaffold. The function ofthe scaffold is to direct the growth of cells mi- trating from surrounding tissue (ssue conduction) or the growth of cll seeded within the porous structure of the scaffold. The scaffold must therefore provide a suitable substrate for cell attach- ‘ment, proliferation, differentiated function, and, in certain cases, cell migation. These eitical re~ quirements can be met by the selection of an appropriate material from which to construct the scaffold, although the suitability of the scaffold may also be affected by the processing technique. Many biocompatible materials can be potentially used to construct scaffolds. However, a biodegradable material is normally desired because the role of the scaffold is usually only a tem- porary one. Many natural and synthetic biodegradable polymers, such as collagen, poly(«-hy- droayesters), and poly(anhydrdes), have been widely and successfully used as scaffold materials due to their versatility and ease of processing (Thomson et al, 1995). Many researchers have used poly(a-hydroxyesters) as starting materials from which to fabricate scaffolds using a wide variety of processing techniques. These polymers have proved successful as temporary substrates for a number of cll types, allowing cell attachment, proliferation, and maintenance of differentiated fanetion. Poly(a-hydroxyesters), such as polyt-latic acid) (PLLA), poly(lactic-co- glycolic acid) (PLGA) copolymers, and poly(gyeolc acid) (PGA), are linea, uncross-inked polymers. These materials are biocompatible, degradable by simple hydrolysis, and are Food and Drug Adminis- tration (FDA) approved for certain clinical applications ‘The major requirement of any proposed polymer processing technique is not only the lization of biocompatible materials, but thatthe process should in no way affect the biocomp: bility ofthe polymer. The processing technique should als allow the manufacture of scaffolds with controlled porosity and pore size, both important factors in organ regeneration. A highly porous scaffold is desirable to allow cell seeding or migration throughout the material. Pore size plays a 1 em) polymer scaffolds, but the coveral size ofthe pores is smaller. Freeze drying has also been applied in another fashion to fabricate porous polymer foams for use in drug delivery applications (Hsu eral, 1997). Scaffolds using this technique have been made with several different types of polymers, including PLLA, PLGA, PGA, and a PLGA/ poly(propy- lene fumarate) (PPE) blend, The desired polymer is dissolved in glacial aetic acid or benzene and frozen to 10°C. The frozen solution is then freeze dried in alyophilizer fora week to remove the solvent. The type of polymer used has a large effect on the morphology of the resulting foams PLGA/PPE foams typically have leaflec morphology whereas PLLA, PLGA, and PGA foams have a capillary morphology. The typeof polymer used also plays a large roe in the release of drugs em- ‘bedded inthe polymer mattix. PLGA/PPF foams release drugs ata much faster rate than do foams rade with PLGA. The lower molecular weight of the PPF allows more penetration of water into the mattix, which causes greater diffusion of drug out of the foam. Polymer foams made in this fashion, however, may not be well suited for tissue engineering applications, due to thei closed ‘pote morphologies and arc limieed to drug delivery use. PHASE SEPARATION Phase separation has been utilized in a novel altemative method of scaffold manufacture, which has been developed primarily to address the problem of drug delivery (Lo et al, 1995). The ability to deliver bioactive molecules from a degrading polymer scaffold to cells within or sur- rounding the scaffold isan atractive one because it could potentially allow manipulation of tissue growth and cell function. In order to achieve chs goa, the scaffold manufacturing process must [end iself vo incorporation of bioactive molecules and must not cause any loss of drug activity due to exposure to harsh chemical or thermal environments. Porous PLLA and poly(phosphocstes) scaffolds with small hydrophilic and hydrophobic bioactive molecules have been manufactured us- ing the following phase-separation technique. First, the polymer is dissolved in a solvent (molten phenol or naphthalene) a 2 low temperature (55°C for phenol and 85°C for naphthalene). The bioactive molecule is then dissolved or dispersed in che resulting homogeneous solution, whichis then cooled in a controlled fashion uni! liquid-liquid phase separation is induced. The resulting21 Polymer Scaffolds bicontinuous polymer and solvent phases are then quenched to create a «wo-phase solid, The 90- lidified solvent is then removed by sublimation, leaving a porous polymer scafold with bioactive ‘molecules incorporated within the polymer. Drug release experiments have shown thatthe activ- ity of small bioactive molecules is not adversely affected by the chemical and thermal conditions utlied in cis phase-separaton technique. This technique has proved useful a 2 means of incor- porating small molecules into polymer scaffolds. Hoteeve, incorporating and releasing drugs with large protein structures without loss of activity remain a challenge. POLYMER/CERAMIC COMPOSITE FOAMS Bone regeneration presents some interesting and unique challenges to engineers and scientists interested in scaffold design. Firs, because almost all bone defects are irregularly shaped, any pro- posed scaffold processing technique mus be sufficiently versatile wo allow the formation of porous polymer-based materials with irregular three-dimensional shapes. Second, the scaffold must have high strength to replace the structural Function of bone temporarily until i is regenerated. Al- though poly(a-hydroxyestes) have been used in a solid form for many orthopedic applications, the compressive strength of foam scaffolds constructed of these materials rapidly declines with ine creasing porosity (Thomson ct a, 1995). In order to improve the mechanical properties ofthese foams, short hydroxyapatite bers were incorporated into the poly(a-hydroxyester) framework in ord to reinforce the scaffold (Thom- son et al, 1998). A melt-molding technique such as the one previously described would be de able asa means to produce a three-dimensional scaffold. Unfortunately, mele molding is not suit ced to the manufacture ofa composite foam. The reinforcing effect of fibers within a polymer matrix is effective only ifthe fibers can be evenly distributed throughout the polymer such that the de- sree offiber-polymer contact is maximized and fiber~fiber contact s held ta minimum, Because achieving an even fiber distribution by attempting to mix «wo solid phases (polymer and fiber) pri- or to molding is extremely dificult, a solvent-cating technique is employed. Hydroxyapatite short fibers and a porogen (either gelatin microspheres or sal particles) are fist dispersed in a PLGA / dichloromethane solution. The esult, afte solvent evaporation and dry. ing, isa composite material consisting of hydroxyapatite fibers and a porogen embedded in a PLGA polymer membrane. The required three-dimensional shape is then achieved by compression mold- ing the composite material. Subsequent leaching of the porogen then leaves a porous composite scaffold of PLGA reinforced with shore hydroxyapatite fibers Fig. 21.5). Within a range of fiber contents, these scaffolds have superior compressive strength compared to nonreinforced PLGA scaffolds ofthe same porosity. “Another process thar utilizes hydroxyapatite to reinforce PLGA foams involves the use of par- ticulate hydroxyapatite rather than the use of fbers (Devin etal, 1996). This technique utilizes an emulsion to create the porous scaffold. The NaCl and the particulate hydroxyapatite are ist suspended in a PLGA solutiotvin chloroform. An aqueous PVA solution is then added tothe sus- pension to form an emulsion. The emulsion is cast into cylindrical molds and then vacuua dried. ‘The dried matrices are then submersed in water to leach out the sat and form the porous scaf- folds. The concentration of the PVA solution and the diameter of the salt crystals both control the diameter ofthe pores formed, which range from 18 ro 150 yum in diameter. Increasing the weight- percent of hydroxyapatite particles in che intial polymer suspension increases the compressive strength of the inal matrix. The compressive properties of the composi scaffold are of the same ‘order of magnitude as those of cancellous bone. An alternative method to formulate polymer/ceramic composites has been proposed using a novel phase transition technique (Zhang and Ma, 1999). In this process, hydroxyapatite powder isadded toa PLGA/dioxane solution. The mixtures frozen fo several hous to induce phase sep- aration and then feeze dred to sublimate the solvent. The formed composite foams exhibit an in- terconnected irregular pore morphology with a polymer/hydroxyapatite skeleton. The compres- sive strength of these foams i significantly higher than that of foams made from pure PLGA. The porosity, pore size, and pore structure can all be controlled by changing the polymer concentra- tion, hydroxyapatite amount, solvent rype, and phase-separation temperature, Composite foams with porosity of up vo 95% and pore siz in the range of 30 to 100 jum can be fabricated with this method. 259260 Fig. 21.5. Scanning electron pho- tomicrographs of highly porous composite scaffolds constructed of PLGA and hydroxyapatite shor fibers using a solvent-cast- ing and melt-molding technique. Sodium chloride particle of size ranging from 300 to 500 um were used as a porogen with an original weight-percent of (a) 70% and (b) 90%. Reprinted from Biomaterials 19, 8.C. Thom son, M. J. Yaszemsti, J. M. Powe ers et al, Hydroxyapatite fiber reinforced — poly(alphahydroxy ester) foams for bone regenera tion, pp. 1935-1943. Copyright 1996, with permission from Else vier Science. Thomson etal IN SITU POLYMERIZATION ‘All ofthe polymer-processng techniques discussed thus far have described methods to man- ufactute prefabricated scaffolds, which may then be used to regenerate che appropriate tissue. Pre- fabricated scaffold ae suitable for most tissues engineering applications; however, there are many instances in orthopedic surgery when mechanical seabiity must be restored immediately. In such cases, a nondegradable bone cement, usually poly(methyl methacrylate) (PMMA), is used co fill the bone defect and provide mechanical stability. A degradable, poly(propylene fumarate)(PPF)- based bone cement has been developed to provide the same Function but on a temporary basis (Yaszemski eral, 1996; Peter eral, 19994). PF isan unsaturated linear polyester and isa viscous liquid at room temperature. It can be cross-linked atthe time of surgery to form solid degrad- able bone cement via an addition polymerization with N-vinyl pyrrolidone (N-VP). As the cross- linking reaction proceeds, the PF is transformed from a viscous liquid to a puttylike state before finally solidifying, During its liquid and putty states, the cement can be injected or molded into the bone defect. Is therefore well suited for this application because many bone injuries resultin defects thac are relatively inaccessible without furcher surgical exposure and are geometrically ill21 Polymer Scatfolds defined, Mose addition polymerization reactions are exothermic and generate large quantities of heat. In the cae of PMMA, which is polymerized in stu, this is sufficient vo cause some local ts- ste necrosis. In contrast, much less hea is generated by the cross-linking reaction between DPF and EVP (Peter etal, 19998), and no local tissue necrosis has been noted in in vivw studies (Yaszemski eral, 1996). Two other components are incorporated into the PF cement at the time ‘of cross-linking. The firs is NaCI, which leacheS out to provide pores into which new bone can ‘grow, and the second is f-tricalcium phosphate, which is an osteoconductive material that facii- tares new bone growth. Incorporation of B-tricalcium phosphate alo enhances the mechanical properties ofthe cement. By using high-molecular-weight PPF and incorporating B-trcalcium phosphate, the mechanical properties ofthe scaffold can be closely matched to that of human tra becular bone (Peter e¢al, 1997, 1998). In this manner, temporary osteoconductive scaffold may be formed in stu to replace the mechanical function of bone until new bone, stimulated to form in the scaffold pores, can assume this structural role. CONCLUSIONS ‘The diversity of organ structure and function is such thatthe design requirements of scaffolds for tissue engineering are specific to the tissue of interest. 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