CLINICAL ORTHOPAEDICS AND RELATED RESEARCH
Number 448, pp. 234–239
© 2006 Lippincott Williams & Wilkins
RGD-tethered Silk Substrate Stimulates the Differentiation
of Human Tendon Cells
T. Kardestuncer, MD*; M. B. McCarthy, BS*; V. Karageorgiou; PhD†; D. Kaplan, PhD†; and
G. Gronowicz, PhD*
Tendon reconstruction surgery often requires healing of the
tendon to bone. The development of a more rapid and strong
interaction at the tendon to bone interface would be invaluable to patients having orthopaedic surgery. Therefore, our
rationale was to modify sutures so that they would be anabolic for tendon to bone healing. It has been shown that silk
stimulates bone formation in osteoblast cultures. In the current study, we tested the ability of silk and silk-RGD (arginine-glycine-aspartic acid) to stimulate human tenocyte adhesion, proliferation, and differentiation. A 1.3-fold increase
in tenocyte adhesion was found on silk-RGD compared with
tissue culture plastic. By 72 hours, proliferation had increased on all substrates but was particularly enhanced on
silk-RGD compared with the control. At 6 weeks, Northern
blot analysis of decorin and Type I collagen mRNA levels
showed a 2-3-fold increase in message levels on silk-RGD and
silk compared with tissue culture plastic. The data suggest
cultured human tenocytes adhere, proliferate, and differentiate on silk and silk-RGD substrates. A suture material,
such as silk, decorated with RGD, may have the potential to
facilitate tendon-bone healing with widespread applications
in tendon reconstruction surgery.
tion and return to activity, and may compromise the final
clinical outcome. To create a more rapid and strong interaction at the tendon to bone interface would be invaluable
for patients. Therefore, our rationale was to modify sutures
so that they would be anabolic for tendon to bone healing.
Sofia et al showed that silk stimulates bone formation in
osteoblast cultures.20 The question we posed in this study
was, can silk also stimulate human tenocyte adhesion, proliferation, and differentiation?
To address this clinical problem, some investigators
have attempted to enhance the rate and strength at which
tendon heals in a bone tunnel. The addition of bone morphogenic proteins (BMPs) into the tunnel has been shown
to induce new bone formation2,14,22 and provide greater
attachment of tendon to bone in dogs.16 In another study,
mesenchymal stem cells used to coat the tendon grafts in
rabbit anterior cruciate ligament (ACL) reconstruction
showed substantially higher load to ultimate failure and
stiffness of the bone-tendon construct at 8 weeks.11 These
advances, however, have some disadvantages. Bone morphogenic proteins are expensive, and routine use in the
clinical setting could be cost prohibitive. The described
techniques can be cumbersome and require the addition of
a wrap or sponge onto the graft. In contrast, suture may be
a useful vehicle for growth factor or anabolic peptide delivery because surgeons routinely use suture during tendon
reconstruction surgery. The suture typically is woven
through one end of the tendon graft and then pulled
through the tunnel, therefore leaving a portion of the suture inside the bone tunnel.
Our goal is to develop novel sutures that would promote
healing at the repair site in tendon reconstruction surgery.
The rationale for this study was first to find an appropriate
material that is capable of stimulating osteoblast and tenocyte proliferation and differentiation. Silk, from silkworms
and spiders, has been shown to be a strong biomaterial
capable of being made into sutures, scaffolds, and
sponges.1 They also have been shown to induce bone formation.20 Furthermore, silk decorated with BMP-219 and
Soft tissue repair and reconstructions of tendons and ligaments require healing of tendon in a surgically created
bone tunnel or suturing with anchors to attach the tendon
to bone. During the early healing process, the repair is
weakest at the attachment site between tendon and bone.15
A weak, bone-tendon interface prevents early rehabilitaReceived: May 2, 2005
Revised: October 12, 2005; November 11, 2005; December 21, 2005
Accepted: January 9, 2006
From the *Department of Orthopaedics, University of Connecticut Health
Center, Farmington, CT; and the †Department of Biomedical Engineering,
Tufts University, Medford, MA.
One or more of the authors (DK, GG) have received funding from the
National Institutes of Health (DE13405); we have no commercial associations.
Correspondence to: Tarik Kardestuncer, MD, 199 Massachusetts Ave., Apt.
1102, Boston, MA, 02115. Phone: 617-948-1600 or 860-680-7670; E-mail:
[email protected].
DOI: 10.1097/01.blo.0000205879.50834.fe
234
Number 448
July 2006
RGD (silk-RGD)4,20 further stimulates bone formation in
vitro. Arginine-glycine-aspartic acid (RGD) is the amino
sequence found in many extracellular matrix (ECM) proteins, and is recognized by integrins (cell adhesion receptors on numerous cell types). Integrins mediate the binding
of cells to the ECM and initiate a signaling cascade that
promote cell proliferation, migration, differentiation, and
survival.8,17 Investigators of in vitro studies described the
benefit of RGD in enhancing cell adhesion to biomaterials6 and promoting fibroblast and osteoblast proliferation.19,20
We chose silk as a bioengineered material because it is
easy to use in suture form, can act as a delivery vehicle for
growth factors, and is anabolic to bone formation. Ultimately, if silk also could induce tenocyte proliferation, it
may be useful to accelerate the attachment between bone
and tendon.
We hypothesized that silk-RGD would induce attachment and proliferation and then differentiation of human
tendon cells in culture better than silk and tissue culture
plastic.
MATERIALS AND METHODS
In replicate (three) experiments, we cultured human tenocytes on
each of three substrates (tissue culture plastic as a control, silk,
and silk with RGD covalently bonded) and ascertained attachment and proliferation and several measures of differentiation
(matrix expression). We isolated silk fibers from the silkworm,
Bombyx mori, and then covalently bound RGD to silk. Next, we
collected fresh human tendon discarded from surgical procedures, dissociated the tendon, and obtained tenocytes according
to the protocol of Maffulli et al.12 Tenocyte adhesion and cell
proliferation were analyzed, and tenocyte differentiation was examined by Northern blot analysis of mRNA levels of Type I
collagen and decorin. The Institutional Review Board of the
University of Connecticut Health Center approved collection of
the fresh human tendons.
Silk films were prepared as described previously.1,20 Briefly,
cocoons derived from Bombyx mori silkworms, were boiled in
0.02 mol/L Na2CO3 and 0.3% (w/v) soap solution, rinsed, and
dissolved in 9 mol/L lithium bromide overnight at 37°C to generate a 10% (w/v) solution. This solution was dialyzed, lyophilized, and dissolved in hexafluoro-2-propanol (HFIP) to give a
2.5% (w/v) solution. Silk films were prepared by adding 75 ␮L
of the HFIP solution in each well of 12-well tissue culture plates
(Falcon, Becton Dickinson and Company, Franklin Lakes, NJ).
A 2.5% solution of HFIP was layered into the wells and dried. A
90/10 (v/v) methanol/water solution was added to the silk film
and dried to prevent the film from resolubilizing in the culture
medium.
Covalent coupling of RGD was accomplished by rehydrating
films in phosphate buffered saline (PBS) buffer (0.1 mol/L sodium phosphate, 0.5 mol/L NaCl, pH 6.5) for 30 minutes.20
Carboxyl groups were activated by reaction with 0.5 g/L 1-ethyl-
Silk/RGD Increases Tenocyte Differentiation
235
3-(dimethylaminopropyl) carbdiimide hydrochloride (EDC) and
0.7 g/L N-hydroxysuccinimide (NHS) in PBS for 15 minutes at
room temperature. After activation, silk films were rinsed extensively and treated with 0.1 g/L glycine-arginine-glycine–aspartic
acid-serine (GRGDS) peptide (CalBiochem, San Diego, CA) for
2 hours in PBS. To remove nonspecifically attached peptide,
films were rinsed with PBS and water and air dried. RGD surface
density was characterized as described before and was 24
pmol/cm2.19 In the current study, covalently coupled RGD to silk
is referred to as silk-RGD.
Fresh human hamstring tendon, discarded from orthopaedic
procedures, was cut into small pieces, digested with 2 mg/mL
collagenase for 3.5 hours at 37°C, pelleted, resuspended, and
plated in Dulbecco’s Modified Eagle Medium (Gibco BRL,
Bethesda, MD) with 10% fetal bovine serum and 100 U/mL
penicillin and 100 ␮g/mL streptomycin.3,13 Cells were fed
weekly until confluent, when they were trypsinized and plated at
a density of 10,000 cells/cm2 for all experiments into six-well
(9.6 cm2) Costar dishes (Corning Inc, Corning, NY).
Initial cell adhesion is important for assessing cell growth and
affects cell proliferation rates, therefore an analysis of human
tenocyte attachment to the three substrates was performed. Substrate-coated plates were incubated in 1% bovine serum albumin
to prevent nonspecific binding for 20 minutes and then washed
with PBS. Cells were cultured for 4 hours on the various substrates and were trypsinized with 4 mL 0.01% trypsin-0.5
mmol/L ethylenediaminetetraacetic acid (EDTA) and counted in
a Coulter Counter (Coulter Electronics, Hialeah, FL).
For determining proliferation, tenocytes were cultured for 72
hours, and in the last 4 hours, 5 ␮Ci/mL of [3H]-thymidine was
added. Cell layers were extracted twice for 5 minutes with 10%
trichloroacetic acid and then lysed with 0.5 N NaOH for 10
minutes. Radioactivity was measured in the lysates using a liquid
scintillation counter (Packard Instrument Co, Downers Grove,
IL), and the radioactive disintegrations per minute (dpm) describe the relative amounts of radioactive thymidine that are
incorporated into the DNA, giving a quantitative measurement of
proliferation.
Six weeks was required to obtain substantial tenocyte matrix
synthesis and differentiation as determined by Northern blot
analysis. After 6 weeks of culture, total RNA was obtained by
the thiocynate-phenol chloroform extraction method described
by Chomczynski and Sacchi.5 Equal amounts of RNA were
loaded into each lane of a 1% agarose gel containing 2.2 mol/L
formaldehyde, and transferred to a nylon membrane in 1X standard saline citrate (SSC) via Posiblot (Statagene, La Jolla, CA).
The bound RNA was immobilized in an UV Statalinker (Stratagene), prehybridized, and then hybridized in 50% formamide, 5
X SSC (0.8 mol/L sodium chloride and 0.08 mol/L sodium citrate), 0.1% sodium dodecyl sulfate (SDS), 0.1 mol/L NaPO4, 5
X Denhardt’s solution (Sigma-Aldrich Corp, St Louis, MO), and
100 ␮g salmon sperm DNA/mL at 42°C. These probes were
labeled with [32]deoxy-GTP using random primer nucleotides.
The filter was washed with 2 X SSC/1% SDS at 65°C and
exposed to Kodak (Eastman Kodak, Rochester, NY) XAR-5 film
at −20°C. The density of bands was measured by using Sigma
Scan software (Jandel Corporation, Chicago, IL) and normalized
to the 28S RNA.
236
Clinical Orthopaedics
and Related Research
Kardestuncer et al
Fig 1A–C. Light micrographs (40×) of tenocytes cultured for 2 weeks on three different substrates are shown: (A) silk, (B)
silk-RGD (RGD), and (C) tissue culture plastic (TCP) which served as a control. The characteristic stellate appearance of
tenocytes on RGD is evident. Bar = 10 µm.
All experiments were performed two to three times with at
least four samples per group. Statistical analysis was performed
by one-way analysis of variance (ANOVA), followed by Student’s t test to determine significance between groups. In this
text, significant differences are p < 0.05.
RESULTS
Freshly isolated human tenocytes grew well on silk, silkRGD, and tissue culture plastic as they appeared to attach,
spread, and proliferate on each substrate (Fig 1). The tenocytes had long thin processes resulting in a stellate appearance.10,13 They reached confluence by 2 weeks of culture.
More cells on silk-RGD had the characteristic stellate morphologic features than cells on the other substrates (Figure
1B versus Figure 1A and Figure 1C). By 4 weeks of culture, the tenocytes had aligned themselves parallel to each
other. By 6 weeks, the cell body became more oval shaped
with prominent nuclei, many granules in the cytoplasm,
and long processes lying over neighboring cells. Tenocytes on silk-RGD completely covered the dish and had
the best morphologic features compared with the other
substrates, which suggested silk-RGD had increased tenocyte proliferation.
Cell adhesion assays showed no difference in cell attachment between tissue culture plastic and silk-coated
plates. However, more human tenocytes attached to silkRGD at 4 hours of culture (Fig 2). A 1.3-fold increase
(p < 0.01) in tenocyte adhesion was found on silk-RGD
compared with tissue culture plastic.
Silk-RGD also enhanced cell growth. At 24 hours, we
observed a 2.0-fold increase (p < 0.05) in proliferation on
tissue culture plastic compared with silk or silk-RGD (Fig
3). By 72 hours, proliferation had increased on all substrates but was particularly enhanced by 2.7-fold on silkRGD (p < 0.01) compared with tissue culture plastic,
which also was greater (1.9-fold; p < 0.05) than on silk
alone.
Finally, silk-RGD promoted tenocyte matrix formation
and differentiation. At 2 and 4 weeks of culture, the mes-
sage levels of decorin and ␣1(I) procollagen (COL) were
not apparent or were very faint in Northern blots. However, at 6 weeks, the expression of mRNA levels was
greater on silk-RGD than the other substrates (Fig 4A).
Northern blots were similar in two experiments at 6 weeks.
At 6 weeks, a 3.0-fold increase in the message levels of
decorin was found on silk-RGD compared with tissue culture plastic by densitometric scanning of the bands and
normalization to 28S ribosomal RNA (Fig 4B). A 2.2-fold
increase was observed on silk, compared with tissue culture plastic. The level of message levels for Type I collagen in tenocytes also were greater on silk-RGD and silk
than on tissue culture plastic.
DISCUSSION
The rationale for this study was to find a material that
could accelerate tendon-bone healing in tendon reconstruction surgery. This idea stemmed from previous studies that showed the ability of various exogenous growth
Fig 2. The bar graph shows tenocyte adhesion on silk, silkRGD (RGD), and tissue culture plastic (TCP) as a control.
Tenocyte adhesion increased (*p < 0.01 ) 1.3-fold when plated
on silk-RGD.
Number 448
July 2006
Silk/RGD Increases Tenocyte Differentiation
237
Fig 3. Tenocyte proliferation on three substrates as measured
by the [3H]-thymidine incorporation is shown in distintegrations
per million (DPM) at 24 and 72 hours. Tenocyte proliferation
increased on tissue culture plastic (TCP) at 24 hours compared with silk and silk-RGD (RGD). However, by 72 hours,
culture proliferation was enhanced (*p < 0.05) on silk-RGD
compared with silk (*p < 0.01) or tissue culture plastic.
factors to facilitate tendon healing and to augment bone
ingrowth into the tendon.2,16 Because tendon-bone healing
occurs by bony ingrowth, it is possible healing could be
improved and accelerated by adding a biomaterial, such as
silk and silk-RGD, already known to stimulate bone formation in vitro, to the tendon graft.4,13,20 Our results show
that silk-RGD also promotes the adhesion, proliferation,
and differentiation of tenocytes in vitro, which supports
our hypothesis that silk-RGD is a promising biomaterial
for augmenting tendon-bone healing.
There are several limitations to this study. Showing that
silk-RGD stimulates tenocyte proliferation and differentiation, and bone formation in vitro does not necessarily
prove it will accelerate tendon-bone healing in vivo. In
particular, the limited suture-tissue interaction may not be
adequate to produce an anabolic effect on the tissue, or
there may be other responses to the silk-RGD that do not
promote healing and are not present in an in vitro setting.
Biomechanical forces and the contribution of marrowderived mesenchymal cells are found in vivo and would
influence tendon to bone healing. However, the in vitro
data presented in this study are promising and deserve
additional study in vivo. The idea of designing a suture
with additional biologic benefits in addition to biomechanical support is also novel and should be considered in
tissue engineering. Finally, although we standardized the
concentration of RGD bound to silk, we have not determined an ideal concentration of RGD. This would require
a dose-response study. Despite these shortcomings, the
clinical relevance of our findings has potential widespread
applications especially in tendon to bone healing, and also,
perhaps, in tendon to tendon healing.
For instance, hundreds of thousands of ACL and rotator
cuff tears occur annually in the US population. Various
Fig 4A–B. (A) Northern blot analysis of decorin and Type I
collagen mRNA levels was performed at 6 weeks of tenocytes
culture on silk, silk-RGD (RGD) and control tissue culture plastic (TCP). (B) Densitometric values of the bands normalized to
28S RNA showed an increase of mRNA for decorin and Type
I collagen (COL) in tenocytes cultured on silk and silk-RGD
when compared with the control (TCP).
surgical procedures have been developed to treat these
injuries. For the ACL, the most common reconstruction
includes placing a harvested tendon, such as a hamstring
autograft, through a surgically created bone tunnel to
mimic the orientation of the native torn ligament. For this
repair to succeed, the tendon must heal and integrate
within the bone tunnel. In the case of rotator cuff repair,
healing is extremely unpredictable, and recurrent defects
have been reported as high as 89%.7 Little is known about
the mechanisms of healing of a tendon graft in a bone
tunnel. Rodeo et al15 described a fibrovascular interface
followed by a collagenous attachment between tendon and
bone resulting in a secure attachment. Others14,18 have
shown a direct type of insertion between tendon and bone
with fibrocartilage between the tendon and bone. Because
these processes require approximately 3 months to achieve
a secure interface, the patient must refrain from loading
the reconstructed ligament during the healing process. Any
238
Clinical Orthopaedics
and Related Research
Kardestuncer et al
material that accelerates bone-tendon healing would result
in earlier return to activity and better clinical outcomes.
Based on the results of this study and on its native characteristics, silk may be the ideal material to achieve this
goal.
Although not the focus of this study, another clinical
application of a silk suture could be its use in direct tendon
to tendon repairs such as in the Achilles tendon or in the
flexor tendons of the hand. In the Achilles tendon, investigators have shown the benefits to tendon healing from a
bioabsorbale scaffold which delivered mesenchymal stem
cells to the repair site.23 In the hand, flexor tendon repair
does not always lead to satisfactory results. Suture type,
strength, and number of crossing strands all have been
implicated in the success of the repair.21 Based, in part, on
the findings from this study, silk-RGD suture might provide the opportunity to repair Achilles and hand flexor
tendons with a strong suture.
Bioengineered silk sutures have an advantage as a biomaterial by having very high strength in combination with
excellent elasticity, and an ability to augment the healing
response in soft tissue repair. Silk’s unique mechanical
properties, versatility in processing, ability to be formed
into sutures, sponges, or scaffolds, along with silk’s biocompatibility and slow rates of degradation, suggest this
biomaterial may become very important in medicine.20
Additionally, the ability to decorate silk sutures with
growth factors or bioactive peptides is appealing for potential use in ligament and tendon surgery. Orthopaedic
surgeons are experienced in using suture in soft tissue
repair, therefore a decorated silk suture would serve the
dual function of mechanical fixation and biologic integration in accelerating the healing response.
Our data suggest cultured human tenocytes adhere, proliferate, and differentiate on silk and RGD-decorated silk
substrates. Tenocyte attachment was particularly increased
on the silk-RGD substrate, compared with silk alone or
tissue culture plastic, which gave similar results. These
responses were similar to the response of human osteoblast-like cells to these substrates.20 Initially tissue culture
plastic stimulated proliferation more than the other substrates, but by 72 hours, silk-RGD showed the greatest
amount of tenocyte proliferation. Because tissue culture
plastic was developed to stimulate cell adhesion and proliferation, the finding of the initial increase in proliferation
on plastic was not surprising, however, silk-RGD shortly
surpassed tissue culture plastic in its ability to stimulate
proliferation, suggesting that silk-RGD must signal the
cell to increase its proliferative capacity. In addition, the
differentiation of tenocytes, as shown by Northern blot
analysis of message levels of ECM proteins, also is stimulated by silk and silk-RGD materials so that proliferation
and differentiation are promoted, suggesting silk-RGD is
anabolic for tenocyte growth and development.
Results of this study support the suggestion that tenocytes respond favorably in the presence of silk-RGD. Previously, others have shown silk-RGD can induce bone
formation.9,20 It follows logically that silk-RGD, when
used in the presence of tendon and bone, may help
strengthen the bone-tendon interface. As this interface is
typically the weakest link in tendon reconstruction, any
biomaterial that can augment tendon-bone healing would
be extremely beneficial. The applications include most
soft tissue surgery about the shoulder, elbow, knee, and
ankle.
Acknowledgments
We thank Dr. Barbara Kream at the University of Connecticut
for providing the ␣1(I) procollagen (COL) cDNA probe and Dr.
Marion Young from the National Institutes of Health for supplying the decorin cDNA probe.
References
1. Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC,
Kaplan DL. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials. 2002;23:4131–4141.
2. Anderson K, Seneviratne AM, Izawa K, Atkinson BL, Potter HG,
Rodeo SA. Augmentation of tendon healing in an intraarticular bone
tunnel with use of a bone growth factor. Am J Sports Med. 2001;
29:689–698.
3. Bernard-Beaubois K, Hecquet C, Houcine O, Hayem G, Adolphe
M. Culture and characterization of juvenile rabbit tenocytes. Cell
Biol Toxicol. 1997;13:103–113.
4. Chen J, Altman GH, Karageorgiou V, Horan R, Collette A, Volloch
V, Colabro T, Kaplan DL. Human bone marrow stromal cell and
ligament fibroblast responses on RGD-modified silk fibers. J
Biomed Mater Res. 2003;67:559–570.
5. Chomczynski P, Sacchi N. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Ann
Biochem Exp Med. 1987;162:156–159.
6. El-Ghannam AR, Ducheyne P, Risbud M, Adams CS, Shapiro IM,
Castner D, Golledge S, Composto RJ. Model surfaces engineered
with nanoscale roughness and RGD tripeptides promote osteoblast
activity. J Biomed Mater Res Part A. 2004;68:615–627.
7. Galatz LM, Ball CM, Middleton WD, Yamaguchi K. The outcome
and repair integrity of completely arthroscopically repaired large
and massive rotator cuff tears. J Bone Joint Surg. 2004;86:219–224.
8. Hynes RO. Integrin: versatility, modulation and signaling in cell
adhesion. Cell. 1992;69:11–25.
9. Karageorgiou V, Meinel L, Hofmann S, Malhotra A, Volloch V,
Kaplan D. Bone morphogenetic protein-2 decorated silk fibroin
films induce osteogenic differentiation of human bone marrow stromal cells. J Biomed Mater Res Part A. 2004;71:528–537.
10. Lamberti PM. Biologic behavior of an in vitro hydrated collagen gel
human tenocyte model. Clin Orthop Rel Res. 2002;397:414–423.
11. Lim JK, Hui J, Li L, Thambyah A, Goh J, Lee EH. Enhancement of
tendon graft osteointegration using mesenchymal stem cells in a
rabbit model of anterior cruciate ligament reconstruction. Arthroscopy. 2004;20:899–910.
12. Maffulli N, Ewen SWB, Waterston SW, Reaper J, Barrass V. Tenocytes from ruptured and tendinopathic Achilles tendons produce
greater quantities of type III collagen than tenocytes from normal
Achilles tendons. Am J Sports Med. 2000;28:499–505.
13. Meinel L, Karageorgiou V, Hofmann S, Fajardo R, Snyder B, Li C,
Zichner L, Langer R, Vunjak-Novakovic G, Kaplan DL. Engineer-
Number 448
July 2006
14.
15.
16.
17.
18.
ing bone-like tissue in vitro using human bone marrow stem cells
and silk scaffolds. J Biomed Mater Res Part A. 2004;71:25–34.
Park MJ, Lee MC, Seong SC. A comparative study of the healing of
tendon autograft and tendon-bone autograft using patellar tendon in
rabbits. Int Orthop. 2001;25:35–39.
Rodeo SA, Arnoczky SP, Torzilli PA, Chisa H, Warren RF. Tendon-healing in a bone tunnel: a biomechanical and histologic study
in the dog. J Bone Joint Surg. 1993;75:1795–1803.
Rodeo SA, Suzuki K, Deng XH, Wozney J, Warren RF. Use of
recombinant human bone morphogenetic protein-2 to enhance tendon healing in a bone tunnel. Am J Sports Med. 1999;27:476–488.
Ruoslahti E. Integrins. J Clin Invest. 1991;87:1–5.
Shino K, Kawaski T, Hirose H, Gotoh I, Inoue M, Ono K. Replacement of the anterior cruciate ligament by an allogenic tendon graft:
an experimental study in the dog. J Bone Joint Surg. 1984;66:672–
681.
Silk/RGD Increases Tenocyte Differentiation
239
19. Shu XZ, Ghosh K, Liu P, Palumbo FS, Luo Y, Clark RA, Prestwich
GD. Attachment and spreading of fibroblasts on an RGD peptidemodified injectable hyaluronan hydrogel. J Biomed Mater Res Part
A. 2004;68:365–375.
20. Sofia S, McCarthy MB, Gronowicz G, Kaplan DL. Functionalized
silk-based biomaterials for bone formation. J Biomed Mater Res.
2001;54:139–148.
21. Strickland JW. Development of flexor tendon surgery: twenty-five
years of progress. J Hand Surg. 2000;25:214–235.
22. Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM, Wang
EA. The healing of segmental bone defects, induced by recombinant
human bone morphogenetic protein. J Bone Joint Surg. 1992;74:
659–670.
23. Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ.
Use of mesenchymal stem cells in a collagen matrix for Achilles
tendon repair. J Orthop Res. 1998;16:406–413.