Biomaterials-based tissue engineering and regenerative medicine

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Peer reviewed article

                       Biomaterials-based tissue engineering
                       and regenerative medicine solutions
                       to musculoskeletal problems
                       Myron Spector
                       Tissue Engineering, VA Boston Healthcare System, and Orthopaedic Research Laboratory,
                       Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

                            Tissue engineering and regenerative medicine          tunity to discover cell biological behaviour when
                       offer solutions to a number of compelling clinical         cells grow in the three-dimensional tissue-like
                       problems that have not been adequately addressed           environment. Selected clinical applications may
                       through the use of permanent replacement devices.          also require the implantation of regulatory proteins
                       The challenge will be to select the optimal combi-         such as growth factors. That the action of such
                       nation of a biomaterial scaffold, cells, and soluble       polypeptides released from biomaterials is short-
                       regulators for a particular clinical problem. For          lived has led to recent work wedding tissue engi-
                       many connective tissues of the musculoskeletal sys-        neering and gene therapy. Genes can be bound to
                       tem, with microstructures that reflect the mechan-         certain biomaterial scaffolds to be released in vivo
                       ical environment, it may be more advantageous to           over extended periods (eg weeks) in order to genet-
                       regenerate the tissue in vivo than to fully engineer       ically modify cells in the defect to produce the
                       the tissue in vitro for subsequent implantation. The       desired growth factors. Thus a new role for bioma-
                       porous material to be used as the scaffold to facili-      terials is as a delivery vehicle for genes, as well as
                       tate this regeneration needs to have certain pore          for cells and growth factors. These endeavours are
                       characteristics, chemical composition and mechan-          notable particularly because there is a growing con-
                       ical properties. One approach has been to employ           sensus that the challenge of developing biomateri-
                       substances that serve as analogues of the extracel-        als for tissue engineering, regenerative medicine,
                       lular matrix of the tissue to be regenerated. For se-      and gene therapy exceeds the challenge that was
                       lected indications in which the supply of endoge-          faced in the cell biological work that led to the
                       nous precursor cells is limited it may be more effi-       proliferation of cells in vitro (in such a way that they
                       cacious to employ a scaffold as a delivery vehicle for     retain their phenotypic characteristics) and in the
                       the cells rather than to inject the cells into the de-     genetic engineering that has led to the production
                       fect. Investigations of cell-scaffold interactions in      of growth factors and cloning of their genes.
                       vitro not only offer the opportunity for modifica-
                       tion of scaffold composition and structure to im-               Key words: tissue engineering; regenerative medi-
                       prove the outcome in vivo, but also offer the oppor-       cine; biomaterials; scaffolds; cells; collagen

                            The term “tissue engineering” has now come                 While tissue engineering investigations have
                       to encompass a wide range of strategies employing          generated promising results, it is important to
                       cells, synthetic and processed natural materials,          point out that no “tissue engineering” procedure,
                       tissues, cytokines and genes for the regeneration of       or any other treatment, has yet been successful in
This work was          tissue in vivo or the production of tissue in vitro.       fully regenerating a tissue that does not have the
supported by the       Cell therapies and tissue transplant procedures are        capability to spontaneously regenerate (eg bone).
U.S. Department
of Veterans Affairs    thus now often considered under the rubric of tis-         One such tissue that does not spontaneously
and Geistlich Bio-     sue engineering. In this respect tissue engineering        regenerate is articular cartilage. It has been known
materials, Wol-
husen, Switzerland,
                       is not so much a revolution in reconstructive sur-         for many years that the long-term function of
which also provided    gery but part of the evolutionary process that this        articular cartilage is related to its composition and
the type II collagen   discipline has continuously undergone since its            architecture, and to the associated mechanical
                       inception over 100 years ago.                              properties. Even relatively small defects in the
Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems                                      294

                         articular surface (figure 1), which can commonly               albeit only in a region of the lesion. This indicates
                         occur, will not heal with a tissue resembling artic-           that articular cartilage regeneration is a possibility.
                         ular cartilage, and in some cases no reparative tis-           The challenge, then, is to identify the elements of
                         sue will form in the defect. Left untreated, such              the regeneration process that need to be supplied
                         defects can extend themselves, eventually (over                to a particular defect: cells, matrix, cytokines, or a
                         decades) resulting in degeneration of the entire               combination.
                         joint. A promising observation, however, has been                   It is also important to recognise that despite
                         that under certain circumstances – sometimes                   the absence of full regeneration of a tissue such as
                         those occurring in an untreated cartilage defect –             articular cartilage, many patients report a dramatic
                         regeneration of articular cartilage can take place,            relief of pain as a result of certain treatments. This
                                                                                        has raised fundamental questions about the crite-
Figure 1                                                                                ria for success that should be adopted in the eval-
Sketch of a human                                                                       uation of new procedures. For example, a clinically
knee joint showing
the type of defect in
                                                                                        meaningful outcome might be one that provides
the articular surface                                                                   pain relief for 5 years, and this may be achieved
that can commonly                                                                       through the formation of a tissue that falls short of
occur. Healing of the
defect will not result                                                                  replicating the composition and structure of artic-
in the formation of                                                                     ular cartilage. Thus, is it more appropriate to use
articular cartilage,
and in many cases                                                                       histological or clinical criteria for success of a new
no reparative tissue                                                                    tissue engineering procedure? The problem with
will form in the de-
fect. Such defects in
                                                                                        using the clinical endpoint is that patients may re-
avascular tissue with                                                                   port a relief of symptoms for a few years only to
limited potential                                                                       experience a precipitous decline in their condition.
of the host cells to
participate in a                                                                        There is no reason to expect that there would be a
reparative response                                                                     gradual decline in function that signals potential
represent clinical
challenges that are
                                                                                        problems and thus allows adjustments in the pro-
addressed by tissue                                                                     cedure or for it to be abandoned before large num-
engineering.                                                                            bers of patients are operated on. Fundamental
                                                                                        questions thus remain as to how to gage the suc-
                                                                                        cess of a new tissue engineering procedure.

                         The scientific basis of tissue engineering
                              As with any engineering discipline the work-              knowledge about the interaction of cells with ma-
                         ing goal of “tissue engineering” is the implemen-              trices we will better be able to prepare new scaf-
                         tation of existing knowledge for the creation of               folds to more specifically elicit the responses from
                         a product – tissue [30]. At the same time, the engi-           cells that best suit a tissue engineering application.
                         neering process often provides opportunities for                    The challenging engineering aspect of tissue
                         the discovery of new knowledge, ie the process of              engineering, as with any engineering endeavour, is
                         science. It is becoming apparent that the unique               the judicious use of existing knowledge for the pro-
                         circumstances related to the growth of cells in                duction of a useful product, in this case, tissue.
                         three-dimensional scaffolds in vitro in the course             There are so many physical and biological issues
                         of tissue engineering are revealing aspects of the             related to the production of tissue in vivo or in vitro,
                         phenotypes of a wide variety of cells and insights             and so few hard facts to guide the engineering
                         into cell behaviours that would have otherwise                 process that tissue engineering is much more of
                         escaped view [30]. In this regard, tissue engineer-            a demanding field than other engineering disci-
                         ing is likely to contribute important new knowl-               plines. Moreover, that the risks of failure include
                         edge to cell and molecular biology, drawn from                 death, greatly increase the stakes of tissue engi-
                         the advancement of health care through the pro-                neering pursuits.
                         duction of tissue in vitro or the facilitation of tis-              Tissue engineering can now be pursued be-
                         sue regeneration in vivo.                                      cause of recent advances in enabling technologies
                              One unique aspect of tissue engineering sci-              related to the tissue engineering triad of cells,
                         ence is the investigation of the interactions of cells         matrices, and regulators. Only recently technolo-
                         with absorbable matrices and environmental fac-                gies have been developed that focus on the prolif-
                         tors (eg mechanical loading) that relate to the for-           eration of cells in vitro under conditions that allow
                         mation of tissue. The cell responses to these inter-           maintenance or recovery of the cell phenotype.
                         actions include cell proliferation and biosynthesis            A critical aspect of most tissue engineering strate-
                         of matrix molecules. More recently it has been ob-             gies is the expansion of cell number in culture in
                         served that cell contraction is another important              order to generate the requisite number of cells for
                         aspect of the cell response to scaffolds employed              the production of tissue in vitro or the implanta-
                         for tissue engineering [30]. As we acquire more                tion of cells alone or seeded in matrices for the re-
                                                     S W I S S M E D W K LY 2 0 0 6 ; 1 3 6 : 2 9 3 – 3 0 1 · w w w . s m w . c h   295

generation of tissue in vivo. Many cell types loose        of tissue in vitro and or in vivo. Synthetic and nat-
critical phenotypic traits with increasing time            ural polymers and calcium phosphates have been
in culture. Advances in cell biology allowed the           developed as scaffolds for the engineering of soft
discovery of culture conditions that favour the            and hard tissues. Control of the pore characteris-
proliferation of cells while a) preserving their           tics including pore volume fraction, pore diameter
phenotype, or b) recovering lost phenotypic gene           and pore orientation, as well as the chemical com-
expression post-expansion. There have been many            position of the matrix, has played a critical role in
advances in the control of culture conditions em-          the advance of tissue engineering. Another impor-
ployed for the preparation of tissue engineering           tant enabling technology that has had an impact
constructs. Some of these new developments have            on tissue engineering is the genetic engineering of
come from the work of Swiss investigators [36],            selected cytokines, such as the bone morpho-
including work employing stem cells [4].                   genetic proteins. These growth and differentiation
     One of the most important technological ad-           factors and agents that stimulate biosynthetic
vances enabling tissue engineering regards the             activity are playing important roles in efforts to
production of the porous, absorbable scaffolds that        form a tissue in vitro and to facilitate regeneration
are required to contain the cells for the production       in vivo.

Tissue engineering: historical perspective
     There are too many investigations that have           plates also revealed the importance of having the
served as the antecedents of tissue engineering to         degradation rate of the matrix, controlled by cross-
include in a review. The following provides a brief        linking, match the regeneration rate in a process
summary of just a few [3].                                 referred to as isomorphous replacement. Later
     Perhaps the earliest successful application of        work in this line of investigation [10] showed for
tissue engineering is the implementation of porous         the first time, in a rat model, that an off-the-shelf
collagen-glycosaminoglycan matrices for the in             scaffold could serve better than an autograft in the
vivo regeneration of dermis [39]. This work that           case of treating gaps in peripheral nerve.
led to the term “artificial skin” served as the basis           Other early studies [37] that used the term “tis-
for the subsequent use of these matrices in tubular        sue engineering” investigated the endothelium-
form for the regeneration of peripheral nerve [38].        like cell layer that formed on the polymethyl
The underlying concept was to develop analogues            methacrylate implants in the eye. Later important
of the extracellular matrix of the tissue to be regen-     work demonstrated the ability of cell-seeded ma-
erated. In addition to demonstrating that selected         trices made from a synthetic polymer to form and
analogues could facilitate the regeneration of tis-        maintain a viable cartilaginous tissue of a selected
sues that did not have the capability to sponta-           shape when implanted in an animal model [11].
neously regenerate, these studies showed that tis-         These and other studies formed the basis of a
sue-specific pore characteristics (ie pore diameter        review article [18] that established tissue engineer-
and orientation) were necessary for the optimal            ing as a distinct discipline.
performance. The use of these regeneration tem-

Tissue engineering versus regenerative medicine
     The term “tissue engineering” was initially in-       function, and can profoundly influence the archi-
troduced to describe the technology for producing          tecture of tissue as it is forming. Because the me-
tissue in vitro [18]. More recently the term “regen-       chanical environment during the formation of
erative medicine” has been used to describe the de-        most musculoskeletal tissue in vivo is not well un-
velopment of technology and surgical procedures            derstood, it is not yet possible to recreate such an
for the regeneration of tissue in vivo. There are ad-      environment in vitro during the engineering of
vantages and disadvantages to both strategies. One         most tissues. Another disadvantage of the forma-
advantage of the synthesis of tissue in vitro is the       tion of musculoskeletal tissue outside of the body
ready ability to examine the tissue as it forms, and       is the necessary incorporation of the tissues after
to make certain non-destructive measurements to            implantation. This incorporation requires that the
establish its functions prior to implantation. How-        engineered tissue be mechanically coupled to the
ever, a disadvantage, particularly in the production       surrounding structures. Union of the implanted
of musculoskeletal tissue that must play a load-           tissue with the host organ requires remodelling –
bearing role, is the absence of a physiological me-        degradation and new tissue formation – at the
chanical environment during the formation of the           interfaces of the implant with the host tissues.
tissue in vitro. It is now well established that me-       That remodelling of the implanted tissue is essen-
chanical force serves as a critical regulator of cell      tial for its functional incorporation.
Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems                                       296

                              Thus, for certain tissues (eg musculoskeletal),           both being referred to as tissue engineering.
                         an effective strategy may be to facilitate tissue              Just as they are the three components of tissue,
                         formation in vivo, under the influence of the                  matrix, cells and soluble regulators are the ele-
                         physiological mechanical environment. However,                 ments of strategies to engineer tissue in vivo, or
                         one disadvantage of this approach is that the                  in vitro for subsequent implantation. Decisions
                         regenerating tissue may be dislodged or degraded               as to which elements might be required for regen-
                         by the mechanical forces normally acting at the                eration of tissue in vivo can be guided by an under-
                         site before it is fully formed and incorporated.               standing of the deficits of the natural (ie spon-
                              In most cases a distinction is not made between           taneous) healing processes that prevent regene-
                         tissue engineering and regenerative medicine, with             ration.

                         Scaffolds for tissue engineering and regenerative medicine
                              For most of the decades of the 20th century, bio-         its influence on cell adhesion and the phenotypic
                         materials have played a critical role in enabling the          expression of the infiltrating cells. Moreover, be-
                         fabrication of a large number and wide variety of              cause the objective is the regeneration of the orig-
                         medical implants. Except for a few examples, how-              inal tissue, the scaffold needs to be absorbable. The
                         ever, these were permanent devices meant to fix or             degradation rate of the material generally may be
                         replace the function of tissues and organs. Stainless          determined based on the rate of new tissue forma-
                         steel devices were developed for the fixation of frac-         tion and the normal period for remodelling of the
                         tures and to fix allografts to host bone. Implants             tissue at the site of implantation. Of course, it is im-
                         fashioned from metallic, ceramic and polymeric                 portant to consider the effects of moieties released
                         materials facilitated life-saving procedures in many           during degradation of the matrix on the host and
                         patients (eg vascular prostheses and artificial heart          regenerating tissue. Finally, the mechanical prop-
                         valves) and profoundly improved the quality of the             erties of the biomaterial employed as a scaffold for
                         lives of other individuals (eg joint replacement               tissue engineering are important for providing
                         prostheses). Despite these remarkable successes,               temporary support of applied loading in vivo dur-
                         the new roles for biomaterials in medicine will                ing the regeneration process and for resisting the
                         likely exceed these achievements. The new roles                contractile forces that may be exerted by the seeded
                         include the use of porous, absorbable biomaterial              cells prior to implantation and by cells infiltrating
                         (sponge-like) scaffolds in tissue engineering, regen-          the scaffold in vivo.
                         erative medicine, and gene therapy.                                 There have been numerous reviews on the
                              Scaffolds for engineering bone and the soft tis-          characteristics of the biomaterial scaffolds gener-
                         sues have been synthesised from an array of syn-               ally employed for tissue engineering [1, 17]. Using
                         thetic and natural calcium phosphates and myriad               a specific biomaterial system (a poly(ethylene gly-
                         synthetic (eg polylactic acid and polyglycolic acid)           col)-terephthalate-poly(butylene)-terephthalate
                         and natural (eg collagen and fibrin) polymers. Scaf-           block copolymer), Swiss investigators and their col-
                         folds for engineering tissue in vitro, or to be used as        laborators recently provided an example of how the
                         implants to facilitate regeneration in vivo, need to           composition and architecture of the scaffold can af-
                         have a microstructure and chemical composition                 fect the behaviour of the cells grown in the scaffold
                         able to accommodate cells and their functions. In              [22]. A comprehensive review of biomaterial scaf-
                         this regard a porous structure is generally neces-             folds is outside the scope of this article. Rather, the
                         sary. The required porosity and pore diameter, pore            author will draw from his personal experience em-
                         distribution, and pore orientation, might be ex-               ploying collagen-based biomaterials (figure 2) to
                         pected to vary with tissue type. The chemical com-             address certain issues related to the use of scaffolds
                         position of the matrix is important with respect to            for specific tissue engineering applications.

Figure 2
Scanning electron
micrograph of a
porous collagen-
scaffold employed
for tissue engineer-

                                                                      1 mm                                                         500 mm
                                                    S W I S S M E D W K LY 2 0 0 6 ; 1 3 6 : 2 9 3 – 3 0 1 · w w w . s m w . c h   297

Roles of a scaffold in tissue engineering and regenerative medicine

    There are many roles that a scaffold can play         to maintain their effectiveness during the remod-
in the tissue regeneration process:                       elling phase that ensues a few weeks to months
– The scaffold can serve as a framework to sup-           after the initial repair procedure. The limited
    port cell migration into the defect from sur-         efficacy of the bolus dosing of growth factors may
    rounding tissues; especially important when a         be due to its inherent inability to maintain thera-
    fibrin clot is absent.                                peutic levels of the cytokine for prolonged periods.
– Before it is absorbed, a scaffold can serve as          The transitory effects of bolus dosing of poly-
    a matrix for endogenous or exogenous cell             peptide growth factors are a consequence of
    adhesion, and can facilitate/regulate certain         their relatively short in vivo half-lives (minutes
    cell processes including mitosis, synthesis, and      to hours), the temporal nature of growth factor
    migration. This may be mediated by ligands            signalling on cellular differentiation and metabo-
    for cell receptors (integrins), on the biomate-       lic function, and the fact that many exogenous
    rial and/or the biomaterial may selectively ad-       cytokines do not stimulate endogenous produc-
    sorb cell adhesion proteins.                          tion.
– The scaffold may serve as a delivery vehicle                 Transfer of the gene for a selected cytokine to
    for exogenous cells, growth factors, and genes.       the cells involved in the reparative process using
    This activity is enabled by a large surface           a scaffold as the delivery vehicle is one means
    area for attachment and the possible control          of maintaining therapeutic levels of the protein
    of the density of the agents (ie agents/unit          through the later phases of the cartilage repair
    volume).                                              process [27]. Non-viral vector systems offer the
– The scaffold may structurally reinforce the de-         advantages of low immunogenicity, simplicity of
    fect to maintain the shape of the defect and          vector design, and relative ease of large-scale pro-
    prevent distortion of surrounding tissue.             duction [12, 25]. The major disadvantage of
– The scaffold can serve as a barrier to prevent          this approach is related to the lower efficiency
    the infiltration of surrounding tissue that may       of transfection. However, for some reparative
    impede the process of regeneration.                   processes (eg articular cartilage) even relatively
                                                          small amounts of the cytokine produced by a few
     The potential role of the scaffold as a delivery     transfected cells may be of significant value. This
vehicle for exogenous cells has become increas-           approach has provided promising results in recent
ingly important in a wide variety of tissues and          studies directed toward enhancing bone regenera-
organs in the light of recent advances in the inves-      tion using a collagen matrix as a carrier for selected
tigation of cell therapy for local repair. Injection      genes [2, 14].
of exogenous cells, expanded in number in mono-                Prolonged release (over several weeks or
layer culture, is being studied for the treatment         months) of DNA from an implant is necessary
of defects and degenerative conditions in many            in cases where there is a benefit in transfecting
tissues:                                                  selected cells that only appear at the implant site
– chondrocytes for the repair of defects in artic-        days or weeks post-operatively, and in which there
     ular cartilage on the surface of joints [8],         is a rapid loss of expression in transfected cells or
– intervertebral disc cells for herniated disc [15],      in which transfected cells migrate from the defect
– stem cells into spinal cord lesions [13],               site.
– myoblasts and stem cells for myocardial infarc-              In one recent study, porous gene-supple-
     tion [31], and                                       mented collagen-GAG (GSCG) matrices were
– cells into the retina [33].                             loaded with plasmid DNA coding for the luciferase
                                                          reporter gene, and the effects of cross-linking and
     An alternative to injection of cells is implan-      pH (during gene loading) on release kinetics and
tation a cell-seeded scaffold. As noted above, the        DNA integrity were determined [25]. The optimal
large surface area of porous scaffolds allows the         conditions showed luciferase expression in chon-
delivery of an exceedingly large number of at-            drocyte-seeded GSCG constructs up to 28 days
tached cells, and facilitates the retention of the        demonstrating continuous transfection of articu-
cells at the implant site.                                lar chondrocytes throughout the culture period.
     Another potential role of the scaffold is the        In a prior study investigating release of plasmid
delivery of genes for selected growth factors [25].       DNA from copolymers of D,L-lactide and gly-
The regeneration of tissue may in some circum-            colide, less than 10% of the DNA remained in
stances require the administration of certain ther-       the synthetic polymer construct after 28 days
apeutic factors (eg growth factors). For example,         in leaching studies performed using Tris-EDTA
selected growth factors, given as a single bolus dose     buffer [26]. Other matrix materials may lend them-
at the beginning of the cartilage repair process,         selves to modification for gene-supplementation
have been shown to accelerate the production of a         for more prolonged release of genes.
hyaline-like reparative cartilage matrix [23]. How-
ever, none of these growth factors have been able
Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems                                    298

                         Methods for the production of scaffolds and design rationale
                              Many methods have been used for the produc-               terials designed specifically for tissue engineering
                         tion of porous materials to be used as scaffolds for           scaffolds. Alternatively the driving force for the de-
                         tissue engineering and regenerative medicine.                  sign of scaffolds may be the precision (computer)
                         These include a) the manipulation of fibers into               multi-scale control of material, architecture, and
                         non-woven and woven structures [18], b) incorpo-               cells: solid free-form fabrication technologies.
                         ration of sacrificial pore-forming agents including            This has become possible with the introduction of
                         ice (through freeze-drying [39]; figure 2) and                 a wide array of solid free-form fabrication tech-
                         soluble particles (eg NaCl and sucrose), c) self-as-           niques and apparatus [32].
                         semblying molecules (eg certain peptides [40] and                   As noted above, one design approach has been
                         collagen-hydroxyapatite composites [21]), and d)               to employ materials that can serve as analogues of
                         solid free-form fabrication.                                   the extracellular matrix of the tissue to be engi-
                              The underlying concepts guiding the develop-              neered [39]. This concept recognises that the mo-
                         ment of scaffolds can be predicated on the selected            lecular composition and architecture of the extra-
                         biomaterial or on the method of production of                  cellular matrix displays chemical and mechanical
                         the scaffold. Examples of biomaterials-based ap-               properties required by the parenchymal cells and
                         proaches include 1) use of biomaterials that have              the physiological demands of the tissue. For scaf-
                         been frequently used for other implant applica-                folds for regeneration of bone, this approach has
                         tions (eg PLA-PGA) [18], 2) treated natural extra-             led to the use of natural bone mineral produced by
                         cellular matrix materials (eg anorganic bone [28]),            removing the organic matter of bovine bone [28].
                         3) biomimetics and analogues of extracellular ma-              For soft tissue applications collagen-based bioma-
                         trix (eg collagen-glycosaminoglycan [39] (figure 2)            terials have been employed [39]. There are special
                         and collagen-hydroxyapatite scaffolds [21]), 4) bio-           issues that need to be considered in the selection
                         polymers for nanoscale matrix (eg self-assem-                  of scaffold materials for the engineering of specific
                         blying peptides) [40], and 5) new types of bioma-              tissues.

                         Investigations of cell-scaffold interactions in vitro:
                         contraction of connective tissue cells
                              Investigations of cell-scaffold interactions in           phenotype as the result of expression of the mus-
                         vitro can inform the rationale formulation of scaf-            cle actin isoform, a-smooth muscle actin (SMA),
                         fold composition and structure for improved per-               chondrocytes were examined for their expression
                         formance in tissue engineering and regenerative                of this cytoskeletal protein. This led to a series of
                         medicine applications. These investigations of                 findings that adult canine and human articular
                         cells in three-dimensional scaffolds that may                  chondrocytes and many other connective tissue
                         mimic certain aspects of the natural extracellular             cells and their mesenchymal stem cell progenitor
                         matrix in vivo can also provide insights into, and             express SMA and can contract [29]. These findings
                         discoveries of, cell biology. In this respect, studies         have suggested roles for the contractile behaviour
                         of the behaviour of cells in collagen-GAG ana-                 of connective tissue cells in the control of the ar-
                         logues of extracellular matrix (figure 2) have been            chitecture of the extracellular matrix and in the re-
                         particularly informative. An advantage of this ma-             sponse of the tissue to injury. The contribution of
                         terial system is the ability to alter selected proper-         muscle actin-expressing and contracting connec-
                         ties through cross-linking: mechanical behaviour,              tive tissue cells to the process of dermal wound
                         degradation rate, and alteration of ligands for the            closure has been recognised for three decades.
                         integrins of cells. Prior work has demonstrated                Muscle actin-enabled cell contraction may also be
                         the effects of cross-link density on the mitosis and           playing important roles in many other connective
                         synthesis of matrix molecules by chondrocytes in               tissues including those comprising the muscu-
                         type I collagen-GAG scaffolds with increasing                  loskeletal system: tendon, ligament, meniscus, in-
                         cross-link density [20].                                       tervertebral disc, articular cartilage, and bone.
                              Several years ago, in the course of investiga-                 In the context of tissue engineering and regen-
                         tions on the behaviour of articular chondrocytes               erative medicine, the mechanical stiffness of the
                         in collagen-GAG scaffolds, the observation was                 scaffold is important in resisting SMA-enabled
                         made that the disc-shaped scaffolds were decreas-              cell-mediated contraction that can alter the shape
                         ing in size. The reduction in volume did not                   of the implant and compress the pores. Recent
                         appear to be due to dissolution of the scaffold.               work, however, has demonstrated that the cell-me-
                         Subsequent histological studies demonstrated a                 diated contraction of a scaffold can be employed to
                         reduction in the pore diameter of the matrices                 favour chondrogenesis in vitro [35]. Chondrocyte
                         and suggested a cell-mediated process. Because                 seeded-scaffolds of varying cross-link densities
                         fibroblasts were known to adopt a contractile                  were cultured for 2 weeks to evaluate the effect of
                                                                               S W I S S M E D W K LY 2 0 0 6 ; 1 3 6 : 2 9 3 – 3 0 1 · w w w . s m w . c h   299

Figure 3
Light micrographs
of chondrocyte-
seeded collagen-
scaffolds with cross-
link density increas-
ing from (A) to (D),
following a 2-week
culture period. The
microtomed 7-µm
thick sections paraf-
fin-embedded speci-
mens were stained
with a Safranin-O/
fast green stain
that labels proteogly-                                     100 µm                                                                       100 µm
cans, a major con-         A                                                           B
stituent of cartilage,
red. The arrows in
panels (C) and (D)
show the residual
minoglycan scaffold
(stained green).
It appeared that a
higher rate of resorp-
tion of the scaffold
was associated with
a higher rate of chon-

                                                           100 µm                                                                        100 µm
                           C                                                           D

                         cross-link density on scaffold contraction and              gineering. In this approach scaffolds would have an
                         chondrogenesis. Scaffolds with low cross-link den-          initial pore diameter large enough to facilitate cell
                         sities experienced cell-mediated contraction, in-           seeding and a mechanical stiffness low enough to
                         creased cell number densities, and a greater degree         allow cell-mediated contraction to yield a reduced
                         of chondrogenesis and an apparent increase in the           pore volume favouring chondrogenesis. This ap-
                         rate of degradation of the scaffold compared to             proach may provide a useful alternative to tradi-
                         more highly cross-linked scaffolds that resisted            tional means of increasing cell number density and
                         cellular contraction (figure 3). The results of this        retention of synthesised molecules that promote
                         study suggest the promise of “dynamic pore reduc-           cartilage formation in tissue engineered con-
                         tion” of scaffolds for articular cartilage tissue en-       structs.

                         Chondrocyte-seeded collagen-gag scaffolds for cartilage repair
                              Studies demonstrating the potential benefit of         ACI, the procedure has been introduced into wide-
                         injection of culture-expanded chondrocytes for              spread clinic use [8] with promising symptomatic
                         cartilage repair date back to rabbit studies first per-     relief in many patients [8, 24].
                         formed in the mid nineteen eighties [9, 16]. Sub-                Current efforts in many laboratories around
                         sequent experiments in a canine model [6, 7] found          the world are being directed to determine whether
                         significantly more hyaline cartilage in the auto-           the results of ACI can be improved when the cells
                         logous chondrocyte implantation (ACI)-treated               are implanted as a cell-seeded scaffold rather than
                         group after 3 and 6 months compared to the un-              delivered by injection. One recent series of studies
                         treated control. At 6 months there was a promis-            compared the reparative tissue in chondral defects
                         ing amount of defect filling with articular carti-          in adult dogs implanted with cultured autologous
                         lage-like tissue. However, by 1 year there were no          chondrocytes (CACs) alone, ie ACI [6], and CAC-
                         significant differences among the treated and con-          seeded type II collagen-GAG scaffolds cultured
                         trol (periosteum alone and non-treated defects)             for 24 hours [5] and 4 weeks [19] prior to implan-
                         groups. By 18 months neither complete filling,              tation. The cell-seeded scaffolds yielded a greater
                         nor the restoration of the architecture was found           amount of reparative tissue than the sites im-
                         [7]. Moreover, cartilage surrounding the defect             planted with the CACs alone. The cell-seeded
                         showed degenerative changes, some of which were             scaffolds cultured for 24 hours induced more
                         related to suturing of the periosteal flap. Despite         reparative tissue formation than the injection of
                         the absence of compelling animal findings using             cells alone. However, this tissue consisted of fibro-
Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems                                                           300

                         cartilage and fibrous tissue with virtually no hya-                     this group also demonstrated the same amount of
                         line cartilage. The question remains as to the rel-                     hyaline and articular cartilage as found in defects
                         ative importance of the amount versus composi-                          implanted with the cells alone [19]. Although these
                         tion of the reparative tissue with respect to provid-                   studies on implementing tissue engineering scaf-
                         ing symptomatic relief for individuals with focal                       folds for cartilage repair are promising, there are
                         cartilage defects. Related to this point is the fact                    potential problems and significant expenses asso-
                         that the hyaline cartilage found at sites treated by                    ciated with culturing a cell-seeded scaffold for
                         CACs alone and in the collagen scaffolds did not                        4 weeks prior to implantation. This draws atten-
                         display the architecture of articular cartilage. Of                     tion to the implementation of growth factors to
                         note was that the greatest amount of reparative tis-                    accelerate cell proliferation and matrix synthesis
                         sue was induced by the CAC-seeded scaffold cul-                         in the scaffolds prior to implantation [34].
                         tured for 4 weeks prior to implantation, and that

                         The future of tissue engineering and regenerative medicine
                              Just as several technologies enabled the devel-                    implants employed for strategies to facilitate tissue
                         opment of tissue engineering as a viable discipline,                    regeneration in vivo.
                         new technologies will provide continuation of its                           The proliferation of cells in monolayer culture
                         growth and maturation. One of these emerging                            and their subsequent growth in three-dimensional
                         technologies is the isolation and expansion of stem                     scaffolds for tissue engineering continues to pro-
                         cells and the identification of the signals required                    vide unique opportunities to observe selected cell
                         for their differentiation into specific cell types. An-                 behaviour. Tissue engineering science will thus
                         other related technology is the genetic modifica-                       provide critical new knowledge that will deepen
                         tion of cells in vitro or in vivo. These technologies                   our understanding of the phenotype of many cell
                         will address the difficulties that are often encoun-                    types and this knowledge will likely enable mean-
                         tered in obtaining a sufficient amount of tissue for                    ingful advances in tissue engineering and regener-
                         the isolation of autologous cells.                                      ative medicine.
                              New matrix materials will likely be developed
                         with selected chemical compositions that allow                               Correspondence:
                         them to better serve as insoluble regulators of cell                         Myron Spector, Ph.D.
                         function. Finally, methods will likely be introduced                         VA Boston Healthcare System
                         to control the mechanical environment of the cells                           Boston Campus, Room D1-152
                         in vitro to better regulate their biosynthetic behav-                        Mail Stop: 151 Research
                         iour. Collectively these approaches will enable the                          150 S. Huntington Ave.
                         synthesis of tissue in vitro that better replicates the                      Boston, MA 02130, USA
                         native material and will be of value in preparing                            E-Mail:

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Description: Tissue Engineering the first term in 1987 organized by the National Science Foundation in Washington, raised the biological engineering team in 1988 formally defined as: Applied life sciences and engineering principles and technology, in the normal and correct understanding of mammalian two states under pathological tissue structure and function relationship, based on research, development for the repair, maintenance, promotion of human tissue or organ injury in a variety of functions and forms of biological substitutes a new study.