Bio Material Technology for Tissue

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REVIEW

Biomaterial technology for tissueengineering applications

Yasuhiko Tabata*

Department of Biomaterials, Field of Tissue Engineering,Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho Shogoin,

Sakyo-ku, Kyoto 606-8507, Japan 

Tissue engineering is a newly emerging biomedical technology and methodology to assist andaccelerate the regeneration and repairing of defective and damaged tissues based on thenatural healing potentials of patients themselves. For the new therapeutic strategy, it is

indispensable to provide cells with a local environment that enhances and regulates theirproliferation and differentiation for cell-based tissue regeneration. Biomaterial technologyplays an important role in the creation of this cell environment. For example, the biomaterialscaffolds and the drug delivery system (DDS) of biosignalling molecules have beeninvestigated to enhance the proliferation and differentiation of cell potential for tissueregeneration. In addition, the scaffold and DDS technologies contribute to develop the basicresearch of stem cell biology and medicine as well as obtain a large number of cells with a highquality for cell transplantation therapy. A technology to genetically engineer cells for theirfunctional manipulation is also useful for cell research and therapy. Several examples of tissueengineering applications with the cell scaffold and DDS of growth factors and genes areintroduced to emphasize the significance of biomaterial technology in new therapeutic andresearch fields.

Keywords: biomaterials; drug delivery system; biosignalling molecules; tissue engineering;tissue regeneration

1. SIGNIFICANCE OF BIOMATERIALTECHNOLOGIES IN TISSUEENGINEERING APPLICATIONS

Advanced surgical therapies currently available consistof reconstruction surgery and organ transplantation.Although there is no doubt that these therapies havesaved and improved countless lives, they have severaltherapeutic and methodological limitations. In the case

of reconstruction surgery, biomedical devices cannotcompletely substitute the biological functions even for asingle tissue or organ, and consequently cannot preventthe progressive deterioration of injured or damagedtissues and organs. One of the biggest issues for organtransplantation is the shortage of donor tissues ororgans. Additionally, the continuous and permanentuse of immunosuppressive agents to prevent immuno-logical rejection responses often causes side effects, suchas the high possibility of bacterial infection, carcino-genesis and virus infection. To resolve these issues inthe two advanced therapies, a new therapeutic solutionthat is clinically mild to patients is required.

In this clinical situation, a new therapeutic trial, inwhich disease healing can be achieved based on thenatural healing potential of patients, has been explored.This trial is termed tissue regeneration therapy wherethe regeneration of tissues and organs is naturallyinduced to therapeutically treat diseases by artificiallypromoting the potential of cell proliferation anddifferentiation. To realize this cell-induced regenerationtherapy, there are two approaches. One is celltransplantation where cells with a high potential of proliferation and differentiation are transplanted toinduce tissue regeneration based on their potentials.The other is the therapeutic approach with biomater-ials and technologies. In the latter approach, an in vivolocal environment that enables cells to promote theirproliferation and differentiation is created by makinguse of biomaterials and technologies. If the environ-ment efficiently manipulates the cells inherentlypresent in the body to enhance the biological potentialsof tissue regeneration, cell-induced natural healing of tissues and organs will be achieved without celltransplantation. This approach is called tissue engin-eering. This basic concept of biomaterial-based tissueengineering was originally introduced by Langer &Vacanti (1993). Cell scaffold and biosignalling molecule

J. R. Soc. Interface  (2009) 6, S311–S324

doi:10.1098/rsif.2008.0448.focus

Published online  4 March 2009

One contribution of 10 to a Theme Supplement ‘Japanesebiomaterials’.

*[email protected]

Received 14 October 2008Accepted 26 January 2009 S311 This journal is q 2009 The Royal Society

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delivery technologies with biomaterials have beendemonstrated to create cell environments suitable fortissue regeneration (Saltzman & Olbricht 2002; Bachet al . 2003; Bannasch et al . 2003; Chen & Mooney 2003;Hubbell 2004; Langer & Tirrell 2004; Silva & Mooney2004; Kuo et al . 2006; Leo & Grande 2006; Tabata2008). For the former approach, generally, cells are

transplanted into the body by the bolus injection orinfusion method. However, few cells are retained at thetransplanted site and their grafted rate is very lowbecause of their excretion and death. In addition, thecells infused are hardly accumulated and grafted atthe site to be regenerated. This low grafting rate of cellstransplanted and their consequent poor functions oftencause low therapeutic efficacy of cell transplantation.To overcome these problems, it is necessary to give thecells an environment suitable to their survival andfunctional achievement. Biomaterials play a key role increating the environment for cells. The scaffold topromote the proliferation and differentiation is pre-

pared from biomaterials, while the biomaterial is usedas the delivery carrier of biosignalling molecules as thecell nutrients to biologically activate cells. Com-bination with biomaterials enables an angiogenic factorto efficiently induce in vivo angiogenesis, which givesnutrients and oxygen to the transplanted cells.Biomaterials need to assist the approach of celltransplantation and enhance the therapeutic efficacy.

This new regeneration therapy cannot alwaystherapeutically substitute the reconstructive surgeryand organ transplantation clinically available, and hasadvantages and disadvantages. However, it is clinicallyexpected as the third therapeutic choice. If this tissue

regeneration therapy is realized, it will enable us tocreate new therapeutic strategies as well as increase thetherapeutic choice of clinicians, which consequentlybrings about large therapeutic benefits for patients whohave not been able to receive clinically effectivetherapies. There are three objectives of regenerationtherapy. The first objective is to create a new thera-peutic strategy of surgery and internal medicine, whichis generally well known. The second objective is toenlarge the clinical application of therapies convention-ally available. Conventional surgical therapy is notalways effective in treating patients who are aged orsuffer from other diseases, such as diabetes and

hyperlipaemia, or cannot be applied because thenumber of key cells is small and their potential forproliferation and differentiation is low. In this case, it ispractically possible that combination with the tech-nology and methodology to promote cell-based self-healing potentials improves the therapeutic efficacyeven for patients who have been clinically treated. Thethird objective is to suppress the progressive deterio-ration of diseases. The deterioration and progress of disease conditions are suppressed by artificiallypromoting cell potentials to induce tissue regeneration.For example, in chronic fibrosis diseases, the fibroustissue of excessive collagen fibres and fibroblasts causes

the impairment of natural healing processes at thedisease site. If the fibrosis can be loosened and digested,and additionally the natural healing potential of thesurrounding healthy tissue can be augmented, it is

highly expected that the disease deterioration andprogression can be suppressed in a physiologicallynatural manner.

At present, there is no effective medical therapy forchronic fibrosis diseases, such as lung fibrosis, cirrhosis,dilated cardiomyopathy and chronic nephritis. Forthese diseases, the injured site is normally occupied

with fibrous tissue of excessive collagen fibres and thefibroblasts excessively proliferate. It is possible thatthis tissue occupation causes the physical impairmentof healing processes at the disease site. Even in adults,the natural healing potential for tissue regeneration stillremains, but cannot operate naturally for certainreasons in disease conditions. For example, therefore,if the fibrosis can be loosened and digested to disappearby any method of drug treatment, it is highly expectedthat the disease site will be regenerated and repairedbased on the natural regeneration potential of thesurrounding healthy tissue. This trial is a new andpossible therapy for chronic fibrosis diseases and

defined as ‘tissue regeneration of internal medicine’because of the drug treatment application of internalmedicine (figure 1). This therapeutic approach issimilar to the surgical regeneration therapy where thecell, the scaffold and the growth factors, or theircombination, are surgically applied to the tissue defectfor generation therapy, because both approaches arebased on the natural healing potential of patients. Wehave demonstrated that the controlled release of amatrix metalloproteinase (MMP)-1 plasmid DNA atthe medulla of chronic renal sclerosis induced thehistological regeneration of kidney structure, incontrast to the plasmid DNA solution (Aoyama et al .

2003). The intraperitoneal release of hepatocyte growthfactor (HGF) histologically cured the liver fibrosis of rats with liver cirrhosis (Oe et al . 2003). A biodegradablehydrogel could achieve the controlled release of smallinterfering RNA (siRNA) for transforming growthfactor (TGF)-b1 type II receptor and regenerate andrepair the fibrosis of chronic renal sclerosis ( Kushibikiet al . 2006).

The basic idea of tissue regeneration therapy is totake advantage of the natural healing potentials of patients themselves. Thus, this is applicable for anothertherapy of internal medicine. For conventional cathetertreatment, an aneurysm occlusion with blood clots has

been clinically performed. However, sometimes therecurrence of aneurysm due to clot lysis is clinicallyproblematic. As one trial to overcome the problem,aneurysm occlusion with tissue organization hasbeen achieved by using coils incorporating basicfibroblast growth factor (bFGF; Kawakami et al  .2005). The bFGF release promoted the cell proliferationinside the aneurysm to allow occlusion by the tissueorganized. The tissue regeneration strategy will beapplied to the therapy of internal medicine.

One of the therapeutic advantages is the ability toaccelerate the natural healing of body injury throughpromoted angiogenesis or the infiltration and recruit-

ment of key cells at the injured site. This will enablepatients to shorten the healing period and suppress thedeterioration process of disease even under inflam-mation and infection conditions. A disadvantage of this

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therapy is that, generally, at least a few days arerequired to induce and activate cell-based tissueregeneration. Consequently, it cannot be expectedthat tissue regeneration therapy alone will achieve therapid healing of wounds or diseases. Depending on the

clinical situation, it is necessary for better medicaltreatment to combine the conventional therapies withthe tissue regeneration strategy.

2. FUNDAMENTAL BIOMATERIALTECHNOLOGY AND METHODOLOGY FORTISSUE ENGINEERING-BASEDREGENERATIVE THERAPY

Basically, body tissue is composed of two components:cells and the surrounding environment. The latterincludes the extracellular matrix (ECM) for cell

proliferation and differentiation (natural scaffold) asthe living place of cells and biosignalling molecules asthe nutrients of cells. There are some cases where tissueregeneration is achieved by the single or combinationaluse of the components in an appropriate way. However,since successful tissue regeneration cannot always beexpected only by their simple combination, it isnecessary to biomedically contrive the way to combine.To this end, proper and positive assistance of biomaterial technology will be practically promising.Biomaterials play a key role in designing and creatingsubstitutes for ECM and the drug delivery system(DDS) of biosignalling molecules to enhance their

biological activities. In addition to therapeuticapplications, biomaterials are also useful in the progressof research and development of stem cell biologyand medicine.

As the biomaterials, various synthetic and naturalmaterials, such as polymers, ceramics and metals ortheir composites, have been investigated and used indifferent manners. Among them, biodegradable bio-materials are explained here. From the practical view-point, metals and ceramics except for calcium carbonateand tricalcium phosphate are not biodegradable. On theother hand, some polymers are biodegradable materials(table 1). The word ‘biodegradation’ is defined to be thephenomenon where a material is degraded or watersolubilized by any process in the body to disappear fromthe site implanted. There are two ways of materialdisappearance. First, the main chain of the materialis hydrolysed or enzymatically digested to decrease

the molecular weight, and finally disappears. Second, thematerial is chemically cross-linked to form a hydrogelinsoluble in water. When the cross-linking bond isdegraded to generate water-soluble fragments, the

Table 1. Biodegradable polymers used for tissue engineeringof cell scaffold and biosignalling molecule release.

synthetic polymers natural polymers

poly(L-lactic acid) ( PLLA) collagenpoly(glycolic acid) ( PGA) gelatinpoly(e-caprolactone) (PCA) fibrincopoly(LL-GA)copoly(LL-CA) hyaluronic acida

copoly(LLA-ethylene glycol (EG)) alginatea

copoly(fumarate-EG) chitosan, chitin

aThere are no enzymes in the body to directly degrade thesepolymers. They are washed out by body fluids to disappearfrom the implanted site.

regeneration and repairing of 

chronic fibrosis based on the natural

healing potential promoted by the

drug treatment of internal medicine

DDS

technology

fibrosis tissue

regeneration and repairing of fibrotic tissue

anti-fibrotic therapy of HGF releaseon liver cirrhosis

HGF solution

HGF released

anti-fibrotic therapy of HGF releaseon dilated cardiomyopathy

HGF solution HGF released

HGF: hepatocyte growth factor

fibrotic tissue is loosened or

digested by the drug treatment.

Tissue regeneration at thefibrotic tissue is achieved

based on the natural healingpotential of surrounding

healthy tissue

Figure 1. A new therapeutic strategy for chronic fibrotic diseases based on the natural healing potentials of patients themselves.The potential is assisted and promoted for tissue regeneration by DDS technology.

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fragments are washed out from the site implanted,resulting in the disappearance of material. Syntheticpolymers are generally degraded by simple hydrolysiswhile natural polymers are mainly degraded enzy-matically. The synthetic polymers can be modifiedwith ease to change their chemical composition andmolecular weight, which affect the physicochemicalproperty of the materials. Natural polymers of proteins,polysaccharides and nucleic acids are available. Theirdegree of freedom for property modification is smallwhen compared with that of synthetic polymers, butthey can be chemically modified to produce variousderivatives. The natural polymers are normally used inthe formulation of hydrogels prepared by chemicalcross-linking. Generally, the synthetic polymers arehydrophobic and mechanically strong when comparedwith natural ones; in other words, their degradationrates are comparatively slow. For the purpose of material application to tissue regeneration, theretention of biomaterials implanted in the body oftencauses the physical impairment of tissue regeneration.On the other hand, an appropriate mechanical strength

of materials is also required. Generally, the mechanicalstrength of materials weakens as their degradationbecomes faster. The two opposite properties should bebalanced by material design and combination.

There are five key technologies or methodologiesthat are necessary for biomaterial-based tissueregeneration therapy and the basic stem cell researchthat scientifically supports the future regenerationtherapy of cell transplantation. The first key tech-nology is for the preparation of cell scaffolds to promotecell proliferation and differentiation for in vivo tissueregeneration (figure 2a ). ECM is not only a physicalsupport for cells, but also provides a natural environ-ment for cell proliferation and differentiation ormorphogenesis, which contributes to cell-based tissueregeneration and organogenesis. Generally, it is difficultto naturally regenerate and repair a large-size tissuedefect only by supplying cells to the defective site,because both the cells and the ECM as well as thesurrounding environment are lost. Therefore, to inducetissue regeneration at the defective site, one possibleway is to artificially build a local environment for cells,which is a three-dimensional scaffold of artificial ECMto initially assist their attachment and subsequentproliferation and differentiation, inducing cell-basedtissue regeneration. It is expected that cells residing

around the implanted scaffold infiltrate into the scaffoldand consequently proliferate and differentiate thereinif the artificial ECM is biologically compatible.Biomaterials play an important role in the preparation

presence of membrane to

provide a space for

tissue regeneration

space providing

regeneratedtissue

a space providing

membrane

fibrous tissue

fibrous tissue

biological barrier by cells and

tissue

free

signalling

molecule

target cells

release carrier

of signalling

molecule

modification of 

signalling molecule

with a water-soluble

polymer

cell-specific

recognition

normal cells

diseased cells

regenerated tissue

the scaffold is combined touse with cells and/or growth

factors depending on thesite to be regenerated

scaffold

cell

transplantation

tissue construct

cells and growth

factors

isolation and

proliferation of stem

cells in bioreactor

cell construct

transplantation

cell sheet preparation by

temperature-responding

substrate

conventional

gene transfection

DNA– carriercomplex

gene transfection from

DNA-immobilized

substrates

(reverse transfection)

transplantation

activation of biological

function by gene

transfection

cells transplanted

in-advance angiogenesis in

the site to be transplanted

space for cell-based tissue regeneration and supply of 

nutrients and oxygen to cells by angiogenesis

the cells cannot survive

without any supplies of 

nutrients and oxygendefect

(a)

(i) (ii)

(iii) (iv)

(i) (ii)

(iii) (iv)

(b)

(c) (d )

(e)

bioabsorbable scaffold

Figure 2. Role of biomaterials in tissue engineering-based regeneration therapy. (a ) Biomaterials for cell scaffold to induce in vivotissue regeneration. Bioabsorbable scaffold: (i) without cells and growth factors, (ii) with cells, (iii) with growth factors, (iv) withcells and growth factors. (b) Biomaterials to protect a space and induce angiogenesis for in vivo tissue regeneration.(c ) Biomaterials for DDS of biosignalling molecules (growth factors and genes): (i) controlled release of signalling molecule,(ii) prolongation of signalling molecule lifetime, (iii) absorption acceleration of signalling molecule, (iv) signalling moleculetargeting. (d ) Biomaterials for in vitro cell manipulation to obtain cells and cell constructs for transplantation. (e ) Biomaterialsfor engineering biological functions of cells.

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tissue regeneration. For example, in addition to haemo-poietic stem cells, mesenchymal stem cells (MSC) arepresent in adult bone marrow. It has been elucidatedthat the MSC of adult stem cells have a proliferativeability and an inherent potential to differentiate intoosteogenic, chondrogenic, adipogenic and myocardialcell lineages. Presently, human MSC are isolated and are

commercially available (Pittenger et al . 1999) whileclinical experiments have been begun. If it is possible toclinically use a differentiated type of a patient’s ownMSC, immunological rejection will no longer be aquestion. Many researchers of tissue regenerationtherapy with stem cells, especially MSC, have reportedtheir therapeutic feasibility in tissue regeneration(Leo & Grande 2006; Docheva et al . 2007; Fibbe et al .2007; Picinich etal . 2007; Shanti etal . 2007). In addition,neural stem cells (Hsu et al . 2007; Kornblum 2007) andstem cells isolatable from fat tissue (Strem & Hedrick2005; Gimble et al . 2007; Schaffler & Buchler 2007) havebeen extensively investigated. They can be prepared

from the foetus and adult and have exhibited plasticityfor cell differentiation and are expected to be promisingcell candidates for regenerative medical therapy.Embryonic stem (ES; Mountford 2008) and induciblepluripotent stem (iPS) cells (Yamanaka 2007) havebeen established and expected as cell sources fortransplantationtherapyandtheresearchand developmentof drugs.

However, one of the problems is the shortage of cellsclinically available. Therefore, it is necessary to developa technology or methodology for the preparation of alarge number of stem cells of high quality. For thispurpose, isolation, induction and in vitro culture

technologies of stem cells are required. The fourthtechnology is for the efficient preparation and prolifer-ation of cells (figure 2d ), which are achieved byproviding a cell culture substrate as the artificialECM. The cell scaffold for in vivo tissue regenerationmentioned previously can be used for the purpose of culture substrate. The three-dimensional substrate canbe designed and prepared from biomaterials of cyto-compatibility. From the viewpoint of nutrients andoxygen supplies, research and development of cellculture methods and bioreactors are required (Holtorf et al . 2006; Mironov et al . 2008).

The fifth technology is to genetically engineer cells

for their functional manipulation and basic biology.There are some cases where cells transplanted do notfunction well to induce cell-based tissue regeneration.As one trial to tackle this issue, cells are geneticallyengineered with biomaterials to activate the biologicalfunctions. It is necessary for the genetic engineering of cells to develop a carrier of gene transfection and cellculture system for efficient gene expression. Generally,viral vectors have been scientifically used for genetransfection because of their high efficiency. However,viruses cannot be used to treat patients; thus, non-viralgene carrier biomaterials are required to be developedfrom the clinical viewpoint of cell therapy. This

technology is also applicable for the basic research of stem cell biology and medicine, which gives importantknowledge and results for cell therapy. This com-bination of cell scaffold, space protection and DDS

technologies is practically promising to create anenvironment that promotes the proliferation anddifferentiation of cells for cell-induced tissue regen-eration. Both the culturing and genetic engineering of cells are key technologies to prepare cells clinicallyavailable for cell therapy. Every biomaterial-basedtechnology is important not only to develop the basic

research of stem cell biology and medicine, but also torealize cell-based tissue regeneration therapy.

3. CLINICAL ASPECTS OF TISSUEENGINEERING-BASED TISSUEREGENERATION

Tissue engineering for clinical regeneration therapy canbe classified as either in vitro or in vivo depending onthe site where tissue regeneration or organ substitutionis performed. In vitro tissue engineering involves tissuereconstruction by cell culture methods and organsubstitution with functional cells—termed bioartificial

hybrid organ. If a tissue can be reconstructed in vitro infactories or laboratories on a large scale, it can besupplied to patients when required. For example,human skin fibroblasts are cultured in a collagensponge to prepare an artificial dermis for skin grafting(Kuroyanagi et al . 2001; Kumagai 2002; Ichioka et al .2005; Takemoto et al . 2008). To prepare pulmonaryvein and bone substitutes for human treatment, thecells of blood vessel and bone marrow-derived stem cellsare cultured in the porous scaffolds of tube-shapepolylactide (Shin’oka et al . 2001) and cubic hydroxylapatite (Ohgushi & Caplan 1999). However, it isdifficult to reproduce the in vivo event completely

in vitro by using the basic knowledge of biology andmedicine or cell culture technologies currently avail-able. At present, it is difficult to realize in vitro tissueengineering because the artificial arrangement of abiological environment to induce cell-based tissuereconstruction is practically impossible. Even if athree-dimensional tissue-like construct is preparedin vitro, it is practically difficult to allow the constructto survive and function in vivo after grafting.In addition, the construct does not always connectwith surrounding natural tissue biologically. Thein vivo environment for the construct implanted shouldbe designed. Another application of  in vitro tissue

engineering is the substitution of organ functions by theuse of allo- or xenogeneic cells. The cells are combinedwith an immunoisolation membrane of biomaterials tosubstitute the physiological functions of liver andpancreas ( Falqui et al . 1991; Olle et al . 1996; Yamashitaet al . 2002; Kobayashi et al . 2003; Ehashi et al . 2006).Biomaterials have been investigated to design andcreate a biological environment that can assist theproliferation and differentiation of cells and maintaintheir biological functions.

Distinct from the in vitro tissue engineering, in vivotissue engineering is advantageous from the viewpointof the environment to induce tissue regeneration. It is

likely that most of the biological components necessaryfor tissue regeneration, such as growth factors andcytokines, are naturally supplied by the body. Based onthis advantage, almost all the approaches of tissue

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engineering have been performed in vivo with orwithout biodegradable cell scaffolds. There are severalexamples where in vivo tissue regeneration is achievedby use of the scaffold with or without cells (Tabata2003b, 2005b, 2008). As described previously, if patientsare young and healthy, and the tissue to be repaired hasa high potential to induce regeneration, active and

immature cells infiltrate the matrix of an implantedbiodegradable scaffold from the surrounding healthytissue, resulting in the formation of new tissue.However, additional means are required if patientsare aged and/or suffer from other diseases, such asdiabetes and hyperlipaemia, or if the regenerationpotential of tissue is low as a result of, for example,a low concentration of cells and growth factors. Thesimplest method is to supply a growth factor to the siteof regeneration for cell differentiation and proliferationin a controllable fashion. As described previously, toallow a growth factor of  in vivo instability to efficientlyfunction in the body, it is necessary to make use of the

DDS technology.One of the largest problems in the release technologyof growth factor protein is the loss of biological activityof protein released from a protein-carrier formulation.It has been demonstrated that this activity loss mainlyresults from denaturation and deactivation of proteinduring the preparation process of the formulation(Gombotz & Wee 1998; Tabata 2003b). Therefore,a method to prepare the formulation of protein releasewith biomaterials should be exploited to minimizeprotein denaturation. From this viewpoint, a polymerhydrogel may be a preferable candidate as a proteinrelease carrier because of its biosafety and its high

inertness towards protein drugs. However, it will bepractically impossible to achieve the controlled releaseof protein over a long period of time from hydrogels onlywhen the protein is simply combined in the hydrogels.The protein combined is normally localized in the waterphase inside the hydrogel and the release is generallydiffusion controlled through the water pathway of hydrogels. The release profile is regulated by modifyingthe cross-linking density of hydrogel polymers.However, there is a limitation for the regulation of protein diffusion by the cross-linking density. There-fore, it is practically impossible to achieve proteinrelease for more than a few days. Considering protein-

induced activation of cell functions, protein releasefor at least 7 days or longer is required. Thus, it isnecessary to contrive a method of protein release.A possible approach is to immobilize a growth factor ina biodegradable hydrogel. It should be noted thatchemical immobilization is not suitable for thispurpose, because the chemical reaction to growth factoroften causes a loss of biological activity. Physicalimmobilization is preferable. The immobilized factoris not released by simple diffusion, but only by thesolubilization of the factor in water accompanied withthe generation of water-soluble hydrogel fragments as aresult of hydrogel biodegradation. In such a release

system, the time profile of growth factor release isgoverned only by that of the in vivo hydrogeldegradation. The requirements of a hydrogel polymerfor this release system are to have biodegradability and

the ability to physically interact with protein forimmobilization. In addition, from the viewpoint of therapeutic applications, it is preferable that thepolymer has been in previous clinical use.

There have been several reports on the controlledrelease of proteins (Andrianov & Payne 1998; Fujiokaet al . 1998; Gombotz & Wee 1998; Tessmar & Gopferich

2007). Some hydrogels were effective in enhancing thein vivo biological activity of growth factors to inducetissue regeneration ( Lutolf & Hubbell 2005; Covielloet al . 2007; Van Tomme & Hennink 2007). Based on therequirement described above, we have selected gelatinto prepare a biodegradable hydrogel protein releasecarrier, because it has been clinically used in medicaland pharmaceutical applications and proven to bebiocompatible. As expected, the hydrogel of gelatinimmobilized the growth factor through the physico-chemical interaction between the gelatin and factor.The growth factor could be released from the hydrogelaccompanied with the degradation of carrier hydrogel

(Tabata et al . 1994, 1999; Tabata & Ikada 1999).Gelatin is easily chemically derivatized to change themolecular nature, which is susceptible to physicalinteraction with protein. Using the gelatin hydrogel,the controlled release of bioactive growth factors over atime range of 5 days to three months was possible. Thecontrolled release of various growth factors hassucceeded in the regeneration therapy of various tissues(figure 3). bFGF is one of the angiogenic factors and hasthe ability to enhance wound healing through aninduction of angiogenesis and induce the regenerationof bone, cartilage, skin, nerve and fat tissues (Tabata2007, 2008). When human recombinant bFGF (Fibrast

spray, Kaken Pharmaceutical Co., Tokyo; http://www.kaken.co.jp) was incorporated into a gelatinhydrogel and subcutaneously implanted into a mouseback, significant angiogenic effect was observed aroundthe implanted site, in marked contrast to the injectionof bFGF solution even at higher doses ( Ikada & Tabata1998). Vascular endothelial growth factor (VEGF) isanother angiogenic biosignalling molecule and has beenextensively investigated to demonstrate the potential toinduce angiogenesis in different forms of DDS modifi-cations (Tabata et al . 2000a ; Zisch etal . 2003; Patel et al .2008). In general, however, blood vessels regeneratedby VEGF are fine when compared with those of bFGF

and VEGF often causes tissue oedema. Comparing thepoints,it is preferable to use bFGF for angiogenic therapyfrom the viewpoint of the therapeutic necessity of wideblood vessels.

There are two important objectives of angiogenesisin tissue engineering: the therapy of ischaemic diseaseand ‘in-advance angiogenesis’ for cell transplantation.As a first example, when injected into the ischaemic siteof myocardial infarction (Iwakura et al . 2003) or legischaemia ( Nakajima et al . 2004), gelatin microspheresincorporating bFGF induced angiogenesis to a signi-ficantly greater extent than the bFGF solution. Thisangiogenic therapy for leg ischaemia has been clinically

shown to demonstrate good therapeutic efficacy ( Maruiet al . 2007; figure 4). This is the first report on clinicalangiogenic therapy by using the DDS technology of growth factor without any cell transplantation.

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The granules of gelatin hydrogels incorporating bFGFwere injected into the femoral muscle of the patient’s

ischaemic leg. bFGF is released locally around theinjected site over two weeks and no bFGF in theblood circulation was detected. When compared beforeand after the bFGF treatment, the pain score, the

tissue oxygen and the blood flow were all significantlyimproved. In addition, the walking distance of 

patients treated increased by a clinically significantextent. We have performed the bFGF-induced angio-genic clinical treatment for 25 patients in fouruniversity hospitals in Japan (ages: 27–73; male/female

0

1

2

3

4

5

6

(i)

(a)

(d )

(e)

(b) (c)(i)

(i)

(iii) (v)

(iv) (vi)

(ii)

(ii)

(ii)

   h

  a   i  r   l  e  n  g   t   h   (  m  m   )

0 020 0.2 2.0 20

VEGF injected (mg/mouse)

in a water

-soluble form

in a released

form

*,**

LCX LCX

LADLAD

Figure 3. Examples of tissue regeneration with biodegradable hydrogels for growth factor release. (a ) Regeneration of coronaryartery: (i) bFGF solution, and (iii) diastole, (iv) systole; (ii) gelatin microspheres incorporating bFGF, and (v) diastole,(vi) systole (LAD, left arterior descending coronary artery; LCX, left circumflex coronary artery). (b) Bone regeneration:(i) BMP-2 solution, (ii) hydrogel incorporating BMP-2. (c ) Promotion of hair shaft elongation (*p!0.05 versus water-solubleform; **p!0.05 versus other VEGF concentrations in a released form). (d ) Articular cartilage regeneration: (i) CTGF solution,(ii) gelatin microspheres incorporating CTGF. ( e ) Fat tissue regeneration: gelatin microspheres incorporating bFGF.Currently, approximately 360 collaborations with the release technology of various growth factors are being performed by

clinicians and researchers.

(a) (b) (c)

(d ) (e) ( f )

Figure 4. Examples of clinical tissue regeneration for ischaemic ASO and diabetic foot ulcer of intractable disease withbiodegradable hydrogels for bFGF release. Intractable diseases could be repaired only by the intramuscular injection orimplantation of hydrogel granules incorporating bFGF. bFGF was locally released over two weeks at the site injected to inducein vivo angiogenesis resulting in promoted wound healing. The first clinical case worldwide: (a ) 27 years, male, (b) four weekslater, (c ) 12 weeks later; (d ) 73 years, female, (e ) four weeks later, ( f ) 16 weeks later. Before treatment, the patients could notwalk due to their severe pain. But angiogenic therapy allowed them to walk without any problem.

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ratioZ15/10). Depending on the patients’ conditions,in the case of diabetic foot ulcer, out of seven patients,four have been healed to recover a condition clinicallyacceptable. In addition, the bFGF-induced angiogenictherapy was also effective in accelerating regenerationhealing of the intractable foot ulcer of diabetes andthe defect of periodontal tissue. Regeneration therapyof sternum, fat and meniscus is being proceeded withclinically (table 2).

The sufficient supply of nutrients and oxygen to cellstransplanted is indispensable for their survival andmaintenance of biological functions in vivo (figure 1b).For successful cell transplantation, it is beneficial toinduce angiogenesis in advance throughout the sitewhere cells are transplanted, by using the bFGF releasesystem. This technology of in-advance angiogenesis

efficiently enhanced the grafting rate and improved thebiological functions of pancreatic islets (Balamuruganet al . 2003), hepatocytes (Ogawa et al . 2001), cardio-myocytes (Sakakibara et al . 2002) and kidney cells (Saitoet al . 2003), as well as the engrafting of a bioartificialdermis–epidermis skin-tissue construct (Tsuji-Saso et al .2007). Consequently, the therapeutic efficacy induced bythe cells and constructs implanted was significantlyaugmented compared with that of no angiogenesis. Therelease system enabled the enhanced activity of variousgrowth factors, such as bFGF, TGF-b1 and bonemorphogenetic protein-2 (BMP-2) to induce bone regen-eration and bone healing, as well as to synergistically

promote bone regeneration induced by MSC of bonemarrow (Tabata et al . 2000b). It is well known thatinsulin-like growth factor (IGF)-1 suppresses the apop-tosis of nerve cells. Controlled release of IGF-1 from thegelatin hydrogel inhibited the ageing of acoustic nerve,resulting in suppressed deterioration of hearing hardness(Iwai et al . 2006). This IGF-1 treatment has been startedclinically to demonstrate good therapeutic efficacy(table 2). In addition, the hydrogel system can releasenot only one type of growth factor but also two or moretypes in different concentrations and release profiles.Upon applying a hydrogel incorporating a low dose of either bFGF or TGF-b1 to a bone defect of rabbit skulls,

no bone tissue was regenerated at the defect. However,a synergistic effect on bone regeneration was observed bythe simultaneous release of two factors ( L. Hong,Y. Tabata, S. Miyamoto, K. Yamada, Y. Ikada 2000,

unpublished data). The synergistic angiogenesis of bFGFand HGF was observed (Marui et al . 2005). The plateletcontains a cocktail of autologous growth factors. Thecontrolled release of the cocktail by the hydrogel enabledthe regeneration of bone (Hokugo et al . 2005), kneemeniscus (Ishida et al . 2007) and intervertebral disc(Nagae et al . 2007), in marked contrast with the use of cocktail alone.

4. FURTHER EXPERIMENTAL ASPECTS OFTISSUE ENGINEERING-BASEDREGENERATION THERAPY

The cell scaffolding technology can not only be appliedto the in vivo cell-based tissue regeneration, but canalso assist and promote the basic sciences in vitro

proliferation and differentiation of stem cells. The latteris for the preparation of cells with a good qualityapplicable for cell therapy and the research develop-ment of stem cell biology. To manipulate the prolifer-ation and differentiation of stem cells in vitro, there aretwo scientific and technological approaches: the modifi-cation of culture medium and cell substrate. There havebeen several trials to add various soluble factors in theculture medium to manipulate cell behaviour.Considering that normally most cells cannot surviveand biologically function without attachment on to theculture substrate, there is no doubt that the substrategreatly affects the profiles of cell proliferation and

differentiation. For example, it has been demonstratedthat the direction of cell differentiation can be modifiedby the softness ( Engler et al . 2006) and size ( Nakamuraet al . 2005) of cell substrates and the surface modifi-cation of biosignalling molecules ( Nagaoka et al . 2006;Benoit et al . 2008). In addition, there have been reportson a three-dimensional scaffold that has a stericgradient of bioactive molecule concentration insidethe material ( Yamamoto et al . 2007). It has been wellrecognized that a biological niche is the local environ-ment of stem cells to naturally regulate their prolifer-ation and differentiation in vivo (Arai & Suda 2007).When the molecular mechanism of stem cell niche is

biologically clarified and the key components can beelucidated and used, the combination with the conven-tional cell scaffold will enable stem cells to artificiallymanipulate their potentials for tissue regeneration.

Table 2. Ongoing clinical experiments of tissue regeneration with the release technology of growth factors.

disease or operation growth factor effectno. of facilities

vascular graft surgery for heart bFGF angiogenesis 1arteriosclerosis obliterans (ASO)

and buerger diseasesbFGF angiogenesis 4

diabetic skin ulcer bFGF angiogenesis, dermatogenesis 3periodontal disease bFGF regeneration of alveolar bone 3hardness of hearing IGF-1 inhibition of acoustic nerve ageing 2meniscus injury PRP chondrogenesis 1facial plastic surgery bFGF chondrogenesis, soft tissue regeneration 1chest plastic surgery bFGF regeneration of sternum bone, angiogenesis 1plastic surgery of soft tissues bFGF angiogenesis 1facial nerve paralysis bFGF nerve function recovery 1

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Future development of stem cell biology and substan-tial collaboration with biomaterials technology willopen a new research field of stem cells and consequentlylead to a promising strategy of cell therapy.

DDS technology and methodology play an import-ant role in the basic research of stem cell biology.Biosignalling molecules to regulate the functions and

fate of stem cells have been elucidated. In thisconnection, for the future, the necessity for DDS willbe undoubtedly increased to develop the basic researchof tissue regeneration and consequently realize stemcell-based regeneration therapy. It is practicallyimpossible to allow biosignalling proteins to functionin vivo without DDS assistance. In addition to thegrowth factors, genes have been used for tissueregeneration therapy. There are two future directionsof gene therapy. The first direction is the conventionalgene therapy where a plasmid DNA and adenovirus aredirectly injected to give the therapeutic effect. For genetherapy, it is practically important to enhance the

efficiency of gene transfection. Generally, the efficacy of viral vectors is higher than that of non-viral carriers.However, the viral vectors are not clinically suitablebecause of their toxicity and immunogenicity.Therefore, it is necessary to improve the in vivo efficacyof gene transfection. One of the possible ways is to useDDS technologies. Several gene carrier biomaterials,such as cationic liposomes and cationic polymers, havebeen investigated to enhance the level of geneexpression (Kushibiki et al . 2003; Kushibiki & Tabata2004; Jo & Tabata 2008). In addition, the releasetechnology is also effective in enhancing genetransfection and expression. The release of plasmid

DNA from a biodegradable hydrogel of cationizedgelatin derivative enhanced the level of gene expressionas well as prolonged the time period of expression(Kushibiki et al . 2003; Kushibiki & Tabata 2004). Theinjection of the cationized gelatin hydrogel incorporatingplasmid DNA enhanced the in vivo therapeutic effects toa significantly greater extent than plasmid DNA solutionalone (Kushibiki et al . 2004). The microspheres incor-porating plasmidDNA alsoenhanced the geneexpressionof cells to genetically activate their biological functionsand consequently increased the efficacy of cell therapy.Using the microspheres incorporating plasmid DNA,intracellular controlled release of plasmid DNA was

achieved to enhance the efficiency of gene transfection toa higher level than that of adenovirus transfection. Thecells genetically engineered functioned well to achievehigher therapeutic efficacy (Yamamoto & Tabata 2006;Jo & Tabata 2008). With the recent development of genomics researches, the DNA sequence of the humangenome has been elucidated and disease therapy on thegenetic level will develop in the future. As proteins,genes are also unstable in vivo. Therefore, it is nodoubt that DDS technology will have an important rolein gene therapy.

The second direction is to genetically engineer cellsfor their functional activation, which is applicable for

cell transplantation therapy and stem cell biology.There are some cases where the transplantation of stemcells alone does not always induce a clinically accep-table therapeutic effect. A promising and practical way

to overcome this problem would be to geneticallyengineer stem cells by gene transfection for theactivation of their biological functions. So far, suchcell activation has been tried by using virus vectors(Lai et al . 2008). This trial has been of great success,but the good results cannot be applied to clinicaltherapies because of the virus use. Therefore, it is

necessary to develop a system of non-virus genetransfection. The DDS technology and methodologyare very effective in developing a non-viral system of gene transfection with the efficiency of gene transfec-tion as high as that of the viral system ( Yamamoto &Tabata 2006; Jo & Tabata 2008). In addition to themicrospheres for the intracellular release of plasmidDNA, a new non-viral carrier has been prepared fromcationized polysaccharides. The carrier of gene trans-fection enabled plasmid DNA to enhance the level of gene expression for stem cells with less cytotoxicitythan commercially available cationized liposomes.Gene transfection with the cationized polysaccharide

was effective in enhancing the gene expression of stemcells to genetically engineer the biological function( Jo & Tabata 2008). In addition, the stem cellsengineered showed the efficacy of cell therapy greaterthan the original cells ( Jo et al . 2007). The level of geneexpression by the non-viral carrier was also enhanced bycontriving the methodology of gene transfection culture,such as a reverse transfection method and bioreactorcombination (Okazaki etal . 2007; Nagane et al . in press).Thus, the gene engineering technology with biomaterialsis effective in enhancing stem cell-based regenerationtherapy and also developing the basic research of stemcell biology and medicine. The technology of gene

transfection will be available for non-viral induction of iPS cells and also allow several bioactive substances tointernalize into cells for an investigation of theirbiological functions in stem cell biology. The efficientaction of siRNA by non-viral carriers is effective ingenetically regulating the cell function. The biomaterialcarrier enables siRNA to enhance the silencing effectin vitro and in vivo, resulting in the artificial modifi-cation of cell functions, which is applicable for stem cellbiology and future cell therapy.

5. CLOSING REMARKS

Tissue regeneration therapy—a new therapy based onthe natural induction of tissue regeneration through celltransplantation and tissue engineering—is a thirdtherapy following reconstructive surgery and organtransplantation. To achieve the regeneration therapyby use of tissue engineering technologies, substantialcollaborative research between material, pharma-ceutical, biological and clinical scientists is needed.Even if superior stem cells can be practically obtained,it is impossible to therapeutically treat patients only bytransplanting the cells prepared even combined withthe scientific knowledge of cells and related substances,unless a local environment of cells suitable to promote

the proliferation and differentiation is created andprovided properly. To create the environment, thereis no doubt that the biomaterials and the technologyare needed.

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However, one of the main problems to create theregeneration environment at present is the absoluteshortage of biomaterial researchers who investigate thecell scaffold, the DDS, the protective barrier and cellculture, aiming at tissue regeneration and the biologicalsubstitution of organ functions. Such researchers musthave knowledge in medicine, dentistry, biology and

pharmacology, in addition to material sciences. It isindispensable to educate researchers of an interdisci-plinary field, who have an engineering background andcan also understand basic biology, medicine and clinicalmedicine. One of the representative interdisciplinaryresearch fields is DDS technology, which is alsoapplicable for producing non-viral vectors in thepreparation of genetically engineered cells for regen-eration therapy. The development of non-viral vectorswith a high efficiency of gene transfection for stem cellsis of a high priority.

Tissue engineering technology is not only usedsurgically for tissue defects, but also applied to develop

a therapeutic method for chronic fibrosis diseases bymaking use of internal medical treatment. Tissueengineering is still in its infancy, although someresearch projects have already come close to the levelof clinical applications. The increasing significance of biomaterials for cell scaffolding and DDS in future willhelp progress in basic and applied tissue engineering.We will be happy if this review stimulates readers’interest in the idea and research field of tissueengineering to assist in understanding how importantbiomaterials are to develop the basic research of stemcell biology and medicine as well as realize tissueregeneration therapy.

The ethics committee of Kyoto University and NipponMedical School approved the study protocol. Patient enrol-ment began in February 2005.

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