Introduction
Mesenchymal stem cells (MSCs) have the potential to differentiate into the tenocyte lineage and regenerate dis- eased or injured tendons. Because there is a limited base of research on the tendon lineage, illustrative examples from the bone and cartilage lineage are included to define more clearly the clinical potential of this cell-based approach to medicine. The concluding sections of this chapter consider the emerging data that support the use of allogeneic/universal MSCs as a cost-effective and prac- tical approach for clinical delivery of MSCs.
Defining Mesenchymal Stem Cells
The name human mesenchymal stem cell (hMSC) gen- erally refers to culture-expanded stem cells that retain the ability to differentiate to several mesenchymal lin- eages. While mesenchymal stem cells are present among marrow stromal cells, not all marrow stromal cells are mesenchymal stem cells. Bone marrow stroma functions to support the hematopoietic stem cells (HSCs) that provide for erythroid, myeloid, and lymphoid cell types needed throughout life. Bone marrow is a complex tissue composed of many cell types including the hematopoietic progenitors and their progeny, mesenchymal stem cells fibroblasts, and endothelial cells, as well as osteoblasts, and adipoblasts.
Early evidence for the multipotential nature of adher- ent marrow-derived cells came from experiments in which animal marrow cells were cultured in vitro, then implanted at ectopic sites in animals. The characteriza- tion of the newly formed tissue demonstrated that the implanted cells could produce several cell types. Many investigations on the nature of adherent marrow stromal cells have been performed since Alexander Friedenstein and his colleagues first demonstrated the osteogenic potential of guinea pig bone marrrow fibroblasts in the
1960s [1,2]. Placement of the cells into diffusion chambers allowed the flow of nutrients, but not migration of host cells. The production of connective tissues following implantation verified that the differentiation capacity lay with the donor marrow cells, not the host cells.This exper- imental approach was extended to rabbit bone marrow cells by Maureen Owen and colleagues, who further characterized differentiation of the cells [3]. Owen also described a limited lineage diagram similar to that pro- posed for hematopoietic stem cells [4]. Arnold Caplan and colleagues, first working with nonhuman mammalian bone marrow-derived cells, and later isolating human MSCs [5], further developed the concept of the connec- tive tissue mesenchymal stem cell and provided a more extensive lineage diagram, encompassing additional tissues [6]. This field has developed through the contri- butions of many investigators, and the reader is referred to several reviews [7–11].
Pittenger and colleagues have developed reproducible isolation methods and characterized a population of human bone marrow-derived cells that are believed to represent the mesenchymal stem cell that persists in the adult [12]. This work provides several important findings.
First, these investigators improved previous isolation and culture conditions and demonstrated the homogeneous nature of the cultured cells. Second, they described in vitro culture conditions that reproducibly promoted dif- ferentiation exclusively to 3 desired mesenchymal lin- eages. Third, they demonstrated that, using human cells of clonal origin, they could recapitulate the same degree of in vitro differentiation as seen in the parental popula- tion, verifying that individual cells held the potential for proliferation and multilineage differentiation (see Figure 31-1, see color insert).
In Vitro Assays
In vitro assays permit an efficient and timely means to determine that MSCs are pluripotent. For example, when 313
31
Tendon Regeneration Using Mesenchymal Stem Cells
Stephen Gordon, Mark Pittenger, Kevin McIntosh, Susan Peter, Michael Archambault, and Randell Young
MSCs are considered for a new preclinical study or for application with a new delivery matrix, the first stage of testing would be in vitro assessments, assuring that these cells differentiate into the appropriate mesenchy- mal lineage. Additionally, in vitro assays allow scientists to explore the factors released by differentiating cells, factors that affect the differentiation process, and gene expression patterns during differentiation.
Osteogenic Lineage
A reproducible system for in vitro osteogenic differenti- ation was established by Jaiswal et al [13]. Optimal cul- turing conditions were established by testing a range of media additives and measuring the best response with assays for alkaline phosphatase, reactivity with antios- teogenic cell surface monoclonal antibodies, modulation
of osteocalcin mRNA production, and the formation of mineralized extracellular matrix. hMSCs derived from second or third passage and cultured in DMEM base media containing 100 nM dexamethasone, 0.05 mM L- ascorbic acid-2-phosphate, and 10 mM b-glycerophos- phate produced optimal results. Figure 31-1 (lower right) shows the osteogenic lineage.
Chondrogenic Lineage
Chondrogenesis has been induced by culturing hMSCs in micropellets in a serum-free DMEM with a supple- ment that included 100 nM dexamethosone, 10 ng/ml transforming growth factor-b3, and other agents [14].
High-glucose DMEM yielded significantly larger pellets than low-glucose DMEM. Within 14 days, differentiated hMSCs secreted an extracellular matrix containing Type Figure 31-1. Multilineage potential of mesenchymal stem cells
(MSCs). Upper left: Human MSCs expanded in monolayer culture. Upper middle: Rabbit MSCs differentiated in vivo into the tenocyte lineage. Upper right: Human MSCs provided stroma support for hematopoietic stem cells growing on a MSC monolayer. Lower left: Adipogenic in vitro differentiation of
human MSCs with oil red O staining of lipid vacuoles. Lower middle: In vitro chondrogenic differentiation of human MSCs with antibody staining for Type II collagen. Lower right: In vitro osteogenic differentiation of human MSCs with staining for alkaline phosphatase in red and von Kossa mineral staining in dark silver. (See color insert.)
II collagen, aggrecan, and proteoglycans. No significant amounts of Type I collagen were observed. Figure 31-1 (lower middle) shows the chondrogenic lineage.
Tenocyte Lineage
In vitro tenogenesis was accomplished by means of a con- struct that consisted of a pretensioned, polyglyconate suture to which cultured MSCs were affixed by way of their contraction of a Type I collagen gel around the suture [15]. This assay was performed in a glass trough culture device in which the pretensioning was provided by a bow-spring formed from stainless steel surgical Kirschner wire (0.89 mm diameter) with a mean restor- ing force of 4.9 ± 0.7 N on the suture. When the MSC- matrix constructs were observed grossly after 40 hours of incubation in the culture device, the matrix had been con- tracted to approximately 30% of the original diameter.
Histological examination of constructs demonstrated an organized structure of elongated cells aligned with the matrix in the direction of tensile load along the longitu- dinal axis. Figure 31-1 (upper middle) shows the MSC tendon construct after it had been implanted at a repair site. Studies on the effect of initial cell-seeding density [16] indicated that constructs seeded at 4 and 8 million cells/mL, as compared to 1 million cells/mL, were more contracted, with greater cell orientation and elongation.
A comparative study of constant versus cyclic tension in the in vitro tenogenesis model was performed by Archambault et al [17]. The constant, static tension was produced by a bowspring formed from Kirschner wire, and the cyclic tension was provided via glass fiber filter tabs on each end of the glass trough loaded onto a Bio- Stretch device. The cyclic loading conditions were 10%
longitudinal strain for 5 seconds at 0.1 Hz for 30 minutes followed by a rest period of 90 minutes and repeated con- tinually for 15 days. Two proteins found in developing tendons, Type VI and Type XIV collagen (undulin), were measured in these in vitro models. Type VI collagen increases occurred about 2 days earlier with cyclic versus static load. Type XIV collagen was present in increased amounts over time for cyclic loading and not present with static loading. Collagen synthesis with cyclic loading was twice the level of synthesis measured for static loading as measured by 3H L-proline incorporation. Figure 31-2 demonstrates histological photomicrographs for in vitro tenogenesis with constant and cyclic loading. Overall, this in vitro system was a good model to demonstrate cellu- lar function and changes in matrix organization in MSC- mediated tenogenesis.
Another in vitro tenogenesis study was designed to investigate the expression of the transcriptional activa- tion genes Eyes Absent 1 and 2 (Eya1 and Eya2) in hMSC-based tendon constructs. The Eya genes are
Figure 31-2. Histology of in vitro tenogenesis of mesenchymal stem cells at 48 hours with constant and cyclic loading. Pho- tomicrographs of H&E stained paraffin sections viewed with fluorescent light at 600x magnification. Photo on left showed
some longitudinal organization of a static loaded MSC con- struct. Photo on right showed a crimp pattern, similar to neo- tendons in fetal tissue samples, with a cyclic loaded MSC construct.
turned on early and transiently in developing mammalian embryonic limbs [18,19]. Static tensioned constructs showed increasing levels of Eya1 and unchanging Eya2 with time. In comparison, cyclic tensioned constructs showed downregulation of both Eya1 and Eya2. These results suggested that in vitro tenogenesis of hMSCs may follow a patterning program similar to fetal development, and analysis of homeobox gene expressions may help to understand this process.
In Vivo Studies with MSCs to Repair Injured Tendons
A 1-cm-long gap injury model in the rabbit Achilles was used to compare a suture alone versus a cell-collagen gel composite contracted onto a pretensioned suture [15].
The cell-composite was formed with autologous MSCs in the same manner as described in the static in vitro tendon studies above. The repair was evaluated at 4, 8, and 12 weeks following surgery. Both structural and material properties of the cell-treated implants were typically about twice the value of controls at all time points. Impor- tantly, the rate of increasing properties with time was greater in the cell-treated implants. Table 31-1 displays material property values at three time points. Qualitative histological examination showed that cell-treated repairs were larger in cross section and better organized than suture alone natural repair tissue.
Another rabbit model was repair of the central third of the patellar tendon, which is a clinical situation aris- ing when using this tissue as a donor graft for anterior cruciate ligament (ACL) reconstruction. Again, the
autologous MSC-composite structure was prepared as described above and evaluated at 3 different seeding densities. The control repair was an unrepaired defect without a suture strut. MSC-treated repair tissues were, on average, significantly stronger and stiffer than natural repair at 12 and 26 weeks postsurgery. No statistically sig- nificant differences were observed among the three seeding densities tested.
Allogeneic/Universal MSCs
Initial preclinical studies using MSCs to treat muscu- loskeletal disorders focused on autologous cells that required a delay between the harvest, isolation, and expansion of MSCs and the implantation for repair and regeneration of damaged tissues. In some clinical situa- tions such as degenerative disorders and injuries requir- ing several weeks of stabilization, autologous MSC therapy would be feasible. Even in these cases, the cost of delivering such a product would be high, due to labor- intensive handling and culture of each individual patient sample and costly release and safety testing.
An improved product configuration would be the use of allogeneic or universal MSCs. Some advantages of an allogeneic MSC product would include: no initial MSC harvest procedure, no delay in applying the treatment, easier tissue handling requirements, no risk of tumor cell contamination from patients with musculoskeletal tumors, low cost of release testing, low final cost of treat- ment, and availability off-the-shelf. McIntosh et al. [20]
have determined that hMSCs appear to be nonimmuno- geneic; therefore, allogeneic MSC therapy is the current focus of preclinical studies (Figure 31-3, see color insert).
Table 31-1. Material properties for treated (T) and control (C) repairs of the Achilles tendon in a rabbit model
4 wk 8 wk 12 wk
Material properties Normal (N = 5) Repair N = 13 N = 13 N = 12 Modulus (MPa) 337.5 ± 205.8 T 53.4 ± 4.9 90.3 ± 10.4 114.4 ± 7.6
C 33.5 ± 7.0 62.2 ± 9.2 67.9 ± 9.8
Stress-maximum 41.6 ± 18.9 T 8.6 ± 0.8 10.5 ± 1.4 15.5 ± 1.1
(N · mm/mm3) C 4.7 ± 1.1 7.2 ± 1.3 8.0 ± 1.2
Strain energy density-maximum 3.9 ± 0.4 T 1.0 ± 0.1 0.8 ± 0.2 1.4 ± 0.2
(N · mm/mm3) C 0.4 ± 0.1 0.5 ± 0.1 0.6 ± 0.1
Strain energy density-failure 6.7 ± 3.6 T 1.3 ± 0.1 1.2 ± 0.2 2.1 ± 0.4
(N · mm/mm3) C 0.7 ± 0.1 0.9 ± 0.1 1.0 ± 0.1
Material properties (mean + SEM) were compared for normal, treated (suture plus MSC con- struct) repair, and control (suture only) repair of a 1 cm gap in the rabbit gastrocnemius tendon.
Modulus represented the increase in stress for an incremental increase in strain in the linear region of the curve; stress-maximum was the largest stress developed during failure testing; strain energy density-maximum was the area under the stress-strain curve to maximum stress; strain energy density-failure was the area under the stress-strain curve to complete loading. All treated means were significantly greater than controls (p < 0.05) except for the strain energy density values at 8 weeks.
In Vitro Evidence Supporting Allogeneic MSCs
The primary in vitro assay to assess the immunologic activity induced by MSCs was the mixed lymphocyte reaction (MLR). This assay is performed by mixing peripheral blood mononuclear cells (PBMCs) from one individual with the PBMCs from a different individual. A subset of T cells in each PBMC population recognize allo- geneic major histocompatibility antigens (Class I and Class II) on cells of the other population and respond by proliferation, which is measured by the uptake of 3H-
thymidine. The assay can be performed as a “one-way”
MLR in which one of the PBMC populations is inacti- vated by irradiation or chemicals to prevent it from pro- liferating. Thus, the one-way MLR can be used to assess the responsiveness of a recipient against potential donors.
The one-way MLR has been used to determine the immunogenicity of allogeneic MSCs. Purified T cells cul- tured with allogeneic irradiated PBMCs proliferated vig- orously, whereas T cells cultured with MSCs (from the same PBMC donor) did not proliferate [20]. Pretreat- ment of the MSCs with IFNg, which is known to upregu- late MHC Class I and Class II alloantigens on the MSCs, did not result in a response to the MSCs. The experiment was repeated using MSCs that were differentiated in the osteoblastic lineage as stimulator cells in the MLR.
Again, there was no proliferative T cell response, indi- cating that immune privilege was preserved in the bone lineage [20].
In vitro tenogenesis studies of Class I and Class II his- tocompatibility antigens were performed using antibody staining. Class I staining was observed in monolayer MSC and tenogenic cultures, while Class II staining was not positive in either culture condition. This demonstrated that the immunogenic potential of MSCs is not changed upon tenogenic differentiation.
In Vivo Evidence of Benefits for Allogeneic MSCs
Several studies have been conducted to determine that implanted allogeneic MSCs do not induce an immuno- logic response and have the capacity to regenerate the appropriate tissue in vivo. The studies described here have used canine and baboon models to demonstrate allogeneic bone formation.
The initial step in studying allogeneic MSC implanta- tion in canines was to prove that the donor-host rela- tionship is immunologically mismatched. Purchasing animals from different vendors in geographically distinct regions was the initial procedure. The canines were then evaluated for satellite markers of dog leukocyte antigen (DLA), which is analogous to human HLA testing. Poly- merase chain reaction (PCR) identifies specific sequences of the key chromosomal segments of class I and class II antigens as clusters of similar and dissimilar histocom- patibility complexes. These results allow interpretation of matched and unmatched pairs of donor and host [21,22].
Finally, the MLR techniques described above can be used to confirm an immunologic mismatch between animals. T cell proliferation of recipient PBMCs cultured with inac- tivated donor PBMCs, a one-way MLR, indicates that the animals are histocompatibility mismatched [20]. In the two canine experiments described below, all three condi- tions were positive, demonstrating that the canines were completely mismatched, allogeneic donor/host pairs.
A
B
Figure 31-3. Histological comparison of allogeneic and autol- ogous MSC bone regeneration. Histological sections from subcutaneous canine study. Samples harvested at 6 weeks, processed through standard decalcified paraffin histology, and stained with modified aniline blue. Off-white color indicated remaining matrix. Blue/brown color indicated new bone for- mation. Vascular ingrowth was also evident. The images repre- sented allogeneic (A) and autologous (B) MSC implants. (See color insert.)
A1
A2
A3
B1
B2
B3
Figure 31-4. (A) Bone formation in autologous MSC-loaded HA/TCP cylinders harvested 10 weeks post-implantation in subcutaneous tissue (MAB stain, Magnification 8¥, 16¥, & 40¥).
Slides stained with modified aniline blue, wherein the orange coloration indicated mature bone and the bright blue staining showed new osteoid. The light blue areas were remaining ceramic matrix. (B) Bone formation in allogeneic MSC-loaded
HA/TCP cylinders harvested 10 weeks postimplantation in sub- cutaneous tissue (MAB stain, Magnification 8¥, 16¥, & 40¥).
Slides stained with modified aniline blue, wherein the orange coloration indicated mature bone and the bright blue staining showed new osteoid. The light blue areas were remaining ceramic matrix. (See color insert.)
In one study [23], a total of 14 canines received sub- cutaneous implants containing allogeneic canine MSCs (cMSCs). Implants were surgically inserted bilaterally with unmatched, allogeneic donors in one limb and autol- ogous cMSCs, from an earlier harvest from the same animal, in the contralateral limb. The implants consisted of cMSCs loaded onto porous hydroxyapatite/tri-calcium phosphate (HA/TCP) cylinders (3 mm O.D. ¥ 6 mm long).
Each limb received 3 implants. Histologic scoring of the explanted cubes, read in a blinded manner, demonstrated that 12 of 14 canines receiving allogeneic implants were positive for cartilage and/or bone at 6 weeks postim- plantation. Similarly, autologous implants had 12 of 14 positive results, indicating no difference in bone tissue formation potential between the allogeneic and autologous MSCs. Histologic and antibody staining for macrophage and monocyte markers demonstrated minimal cell infiltration, equivalent to that of native tissue. Figure 31-3 shows examples of in vivo histologic comparisons between autologous and allogeneic MSC implants. In addition, absence of CD3 staining verified the lack of T cell infiltration into the MSC-ceramic implant. Overall, there was no observed immunologic response to the allogenic MSCs.
A pilot study in baboons was conducted to evaluate allogeneic bone formation in a subcutaneous site.
HA/TCP ceramic cylinders, 6 mm in length and 3 mm in diameter with a central canal, were loaded with either autologous or allogeneic MSCs. The allogeneic MSCs came from donors with complete HLA mismatches in both Class I and II alleles. Cylinders were incubated for 10 hours to allow MSC attachment, and then held at room temperature for 36 hours prior to implantation. After 10 weeks, the cubes were explanted and processed for his- tological analysis. Figure 31-4 (see color insert) shows autologous and allogeneic paraffin histologic sections at three magnifications. All cylinders showed extensive bone formation, whether they contained MSCs from an allogeneic donor or isolated from the animal’s own marrow. No inflammatory response or rejection of the implants was noted.
Another study [24,25] evaluated the ability of allo- geneic canine MSCs to heal a critical-sized 21 mm, osteoperiosteal, segmental defect in the canine femur. The bone was stabilized with a fixation plate, and the cMSCs were loaded onto an appropriately sized HA/TCP matrix.
Healing response was evaluated at 4, 8, and 16 weeks with an N = 4 at each time point. Bone was present in the bone- implant interface as early as 4 weeks following surgery. At 8 weeks, a large bony callus was observed. At 16 weeks, there was a substantial amount of bone formation. These results were compared to a previous study [26], using the same surgical model, that compared empty defects to cell- free and cMSC loaded HA/TCP matrices. The amount of bone formation with allogeneic MSCs was similar to that
observed in the autologous case, both of which were greater than for implants with no MSCs added. In all cases with allogeneic MSCs there was no histological evidence of an immunologic response by the host.
Clinical Applications and Future Directions
Current tendon regeneration studies using MSCs are encouraging. However, additional preclinical research is required before clinical applications can be considered.
Clinical applications for tendon could include: Achilles injuries requiring surgery and long rehabilitation; flexor tendon injuries of the finger, which heal poorly with adhesions and reduced range of motion; and mid-third patellar tendon harvests for ACL reconstruction, which heal slowly and incompletely with long-term donor site morbidity.
Ligament injuries, such as a torn ACL, are a major clin- ical challenge that could be significantly improved with a regenerative treatment approach. Recent studies in a normal and OA goat knees have indicated that MSCs can survive and remain attached to soft tissues in the harsh environment of an inflamed knee joint. Therefore, research has been initiated into regeneration of ACLs.
Research and development will progress sequentially from autologous to universal MSC-based therapy for lig- ament regeneration.
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