2. Mesenchymal cell
Mesenchymal stem cells (MSCs) were generally defined the cells having the capacity to self-renew and maintain
their phenotype.
The International Society for Cellular Therapy (ISCT) has proposed the following minimum criteria for the
definition of the MSCs:
(I) adherence to plastic surfaces under standard cell culture conditions;
(II) the expression of cell surface markers, such as CD90, CD73, and CD105, and the lack of
expression of CD14, CD34, CD45, CD79, or CD19 and HLADR
3.
4.
5. Mechanism
Mesenchymal stem cells (MSCs) are stromal cells that are found in many tissues but can be readily harvested
from adult bone marrow and adipose tissue & culture expanded.
Following intravenous injection, these cells have been shown to home to sites of inflammation or tissue damage.
As MSCs appear to be largely non-immunogenic (due to their low constitutive expression of major
histocompatibility complex I and II proteins, and the lack of T-cell costimulatory molecules) culture-expanded
allogeneic MSCs may represent a practical, cost-effective stem cell therapy that does not have the ethical issues
associated with embryonic stem cells.
Once therapeutically applied, MSC can either act directly by homing to particular anatomical sites after
transplantation and differentiating into specific cell types to locally restore the damaged tissue.
Even more important, MSCs can support tissue regeneration by a paracrine mechanism of action, such as
secretion of multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting
inflammation.
In addition, MSCs lack immunogenicity and possess the ability to perform immunomodulatory functions
6. Differentiation capacity
Multipotent- Can differentiate into
Osteoblasts
Adipocytes
chondrocytes
myocytes
neuron-like cells.
Differentiation capacity decreases with the age of the donor
and the time in culture
8. Mesenchymal cell separation
120-150 ml of
bone marrow
in Heparin
Density
Gradient
separation by
Centrifugation
Total count
and viability is
checked by
cell counter
9. Culture and Induction of MSCs to Osteoblast
DMEM: Dulbecco’s Modified Eagle Medium
FBS: Fetal bovine serum
10. Regenerative Medicine
Two different strategies of cell-based therapy
1. In the first approach, cells are applied to substitute damaged cells
within a tissue to reconstitute its integrity and function. During this
procedure called “cell therapy” a cell suspension is simply injected into
the damaged tissue or into the blood circulation.
2. The second approach called “tissue engineering” is more complex.
Here, cells are combined with a three dimensional matrix to compose a
tissue-like construct to substitute lost parts of the tissue, or even whole
organs.
11. Tissue Engineering
The technique, tissue engineering requires three components :
growth-inducing stimulus (growth factor)
responsive cells, and
scaffold to support tissue formation
12. Stem cell action
•A. Direct repair
•B. Soluble growth factors ,cytokines-Paracrine effect
•C. Immunomodulatory effects
MSCs exhibited potent anti-inflammatory and
immunomodulatory activities both in vitro and in vivo.
MSCs secreting a range of immunomodulators, including
transforming growth factor b, prostaglandin E2, interleukin
10, indoleamine 2,3-dioxygenase.
14. Bone Marrow-Derived Mesenchymal Stem
Cells
In Nut Shell
BMSCs Steps in Tissue Engineering
Taking the stem cell from iliac crest
Culture the stem cells 4-6 weeks lab
Debridement the lesion
Taking tibial periosteum
Suturing the periosteum to the defect,
Implantation of stem cells, completing the suture
Fibrin glue
17. MSCs in Non-Union bone fracture
MSCs have the osteogenic differentiation ability, can secrete a variety of cytokines and promote
angiogenesis, thus accelerate fracture healing.
After traumatic injuries, BMSCs could migrate from blood circulation to the lesion site, and then directly
differentiate locally, and replace the injured cells.
But the majority of the cells at the fracture site are migrated from the adjacent tissues. Therapeutic
amplification of circulating MSCs through their mobilization could also represent a potential therapeutic
opportunity in fracture repair .
Indirectly, BMSCs promote bone healing mainly through the secretion of bioactive molecules and
extracellular membrane vesicles, which induce angiogenesis, regulate inflammation, inhibit apoptosis, and
regulate osteogenesis differentiation.
MSCs are known to secrete angiogenesis related factors include angiopoietin Ang-1 and Ang-2, vascular
endothelial growth factor (VEGF), FGF-2, and hepatocyte growth factor (HGF)-1 [17]. Furthermore,
BMSCs have an anti-fibrotic effect and can limit that fibrosis progression of fracture zone and promote the
regeneration of bone tissue.
18. MSCs in articular cartilage injury
Due to the limited ability of proliferation capacity of chondrocytes, articular cartilage injury often causes progressive
degeneration of the joint and OA, which is a serious health and economic problem.
The current treatment ---- microfracture, uses the body’s own healing abilities to regenerate the chondral surface.
However, the regenerated fibrocartilage often has poor mechanical properties compared with normal cartilage.
The MSC-based autogenous transplantation treatment, since the potential of the MSCs to differentiate into chondrocytes.
Tissue Engineering
(1) during the first operation, a cartilage biopsy is taken from areas of damaged cartilage within the ankle or knee;
(2) chondrocytes are isolated from the biopsy via enzymatic digestion and cultured in 2D monolayer cultures;
(3) Monolayer culture-expanded chondrocytes are seeded on a collagen type I–III membrane;
(4) in the second operation, the cartilage lesion is prepared and the collagen membrane is cut to size, placed in the lesion
and secured with fibrin glue.
Zwolanek et al. describe a novel cell tracking system based on genetic transgenic donor and corresponding cell marker,
and the results showed that MSC could contribute to cartilage regeneration via a progenitor or non progenitor - mediated
mechanism.
19.
20. MSCs in meniscus injury
Meniscus injury in the knee joint is probably the most frequent intra-articular damage.
The typical treatment is a partial meniscectomy or repair, but it can lead to degeneration of articular
cartilage, narrow joints, and early osteoarthritis.
Intra-articular injection of MSCs could be a simple treatment to promote the regeneration of
meniscus.
When MSCs were injected directly into the articular cavity, they could migrate to the lesion site,
directly participate in the tissue repair, and induce the repair of the host through the collateral
secretion, and replace the injured tissue.
Murhpy et al. reported the first study of injection of BMSCs in sheep articular cavity [37], and
observed the obvious repair of cartilage damage in meniscus injury, 6 and 12 weeks after injection.
However, Hong et al. used arthroscopic surgery to repair the meniscus of the posterior articular cavity
with or without BMSCs after meniscus injury, and found that the meniscus and tibial plateau were
not fully integrated, and the efficacy of MSCs treatment group was not significantly different from
that of the control group. They argued that MSCs may differentiate into other tissue cells if they were
not effectively induced to differentiate into specific cell types. Therefore, it is still a challenge to
induce the cells into the meniscus cartilage phenotype in this context.
21. MSCs in the treatment of osteoarthritis
Osteoarthritis (OA) is a major cause of joint pain and loss of mobility in
the elderly, which seriously affects the quality of life and causes huge
social and economic burden.
Many researchers have conducted a series of clinical studies on BMSCs
transplantation to treat OA, and these studies demonstrated that
moderate confidence could be placed on the safety of MSCs therapy for
knee OA, but the confidence in efficacy outcomes is low, mainly due to
limited clinical case number. Therefore, further high-quality studies for
OA with high internal and external validity are still required.
Shi et al. compared the clinical results of platelet-rich plasma (PRP) and
MSCs treatments for osteoarthritis of the knee in a systematic review
and pointed out MSCS provide more significant disease therapeutic
effect
22. MSCs in femoral head necrosis
Numerous clinical methods have been tried, including core decompression (CD) .The presumption is that CD can
reduce the intraosseous pressure and also stimulate stem cell regeneration. But the outcome of CD is variable and is
still controversial.
With the development of non-biological materials, MSCs and tissue engineering techniques, the treatment of ANFH
has been significantly improved recently.
Various studies reported that the efficacy of MSCs transplantation group was significantly better than that of the pure
medullary decompression group.
In study by Dr Phillipe,100 patients with early-stage ANFH were recruited and randomly assigned to BMMSC
treatment or CD treatment only, this intervention was proved to be safe and more effective in delaying or avoiding FH
collapse.
In another study of eight patients with bilateral femoral head necrosis, the researchers performed the medullary
decompression on one side, while on the other side medullary decompression MSCs transplantation. The Harris hip
score (HHS) and VAS score of the MSCs transplantation group were significantly improved, and the results of MRI
quantitative analysis showed a significant decrease in necrosis area.
Consistently, another study found that the group of MSCs had a significantly superior recovery of the early stages of
necrosis.
However, there have been reports of unsatisfactory success rates for end-stage osteonecrosis of the femoral head
(ONFH), even with MSCs.
23. MSCs in intervertebral disc degeneration
Intervertebral disc degenerative is a serious worldwide problem for the aging population.
The apoptosis of nucleus pulposus cells could be the main cause of intervertebral disc degeneration, with a
variety of manifestations, that is, reduced number of the cells, the changes of the mechanical structure,
down-regulated synthesis of matrix components (such as proteoglycan), nucleus pulposus dehydration,
and increased metabolic waste.
MSCs transplantation provides a new therapeutic strategy for promoting proteoglycan synthesis,
decelerating the course of disc degeneration, and stimulating disc regeneration.
In a study, under fluoroscopic guidance, the BMCs were injected into the nucleus pulposus of 26 patients’
with chronic (>6 months) discogenic low back pain. Found the evidence of safety and feasibility in the
non-surgical treatment of discogenic pain using autologous BMCs with durable pain relief (71% VAS
reduction) and Oswestry Disability Index improvements (>64%) through 2 years.
Overall, the BMSCs as a treatment of degenerated intervertebral disc is successful both in an animal model
and in clinical studies; however, there are no long-term follow-up results and the number of reports and the
number of cases are still relatively low.
BMSCs may cause osteophyte formation in the vertebral isthmus when it is released from the nucleus.
24. MSCs in osteoporosis
Osteoporosis is a common metabolic bone disease, characterized by loss of bone mass, bone density
reduction, and bone structure damage, which leads to increased bone fragility and risks of bone fracture.
The exact underlying mechanisms of osteoporosis are still unclear, but a shift of the cell differentiation of
MSCs to adipocytes rather than osteoblasts partly contributes to osteoporosis. It was observed that osteoclast
activity was enhanced, while osteoblast function decreased.
Recently, it is found that the decrease of BMSC to osteogenic differentiation and the increase of lipid
differentiation is an important factor in the pathogenesis of osteoporosis.
BMSCs transplantation can also effectively increase bone mass and density, increase bone mechanical
strength, correct the imbalance in bone metabolism, and increase bone formation, and is expected to provide
a new strategy and method for the treatment of osteoporosis.
The clinical trials of MSCs in osteoporosis have begun; nevertheless, the animal studies have already found
that autograft or allogeneic MSC transplantation can increase the bone mass of animal models of
osteoporosis.
Since osteoporosis is a systemic disease, and the hormone levels and cytokines have changed dramatically, it
is still unclear whether the simple local MSC transplantation can improve these changes in the long-term.
In addition, the bone marrow homing efficiency of MSCs and the long-term survival of MSCs are still
25. MSCs in genetic diseases
Osteogenesis imperfecta (OI)
Osteogenesis imperfecta is a rare congenital bone development disorder characterized by bone fragility, blue sclera,
deafness, and joint relaxation.
Horwitz et al. reported three cases of OI using allogeneic bone marrow cells. Six months after the implantation, the
new bone formation was observed with a reduced frequency of fractures, suggesting that bone marrow cells could be
used to treat OI.
Hypophosphatasia (HPP)
Hypophosphatasia is a rare, heritable, metabolic bone disease due to deficient activity of the tissue-
nonspecific isoenzyme of alkaline phosphatase. The disease is characterized by the disturbance of bone
and tooth mineralization and reduced serum ALP activity.
Tadokoro et al. used allogeneic MSCs obtained from the patient’s father for an 8-month-old patient with
hypophosphatasia, and they observed improved respiratory condition, and de novo bone derived from both
donor and patient cells.
Similarly, Cahill et al. reported an 8-month-old girl with worsening and life-threatening infantile HPP
improved considerably after marrow cell transplantation. 4 months after treatment, radiographs
demonstrated improved skeletal mineralization.
26. MSCs in Pediatric Orthopaedics
Muscular dystrophy is a diverse group of neuromuscular disorders that result in progressive muscle
weakness, muscle atrophy, paralysis, and death in severe cases. The current management of some muscular
dystrophies involves pharmacological suppression of the immune and inflammatory responses.
Unfortunately, beneficial effects of these therapies are only modest and temporary.
Wakitani et al. found that, under certain circumstances, in vitro BMDMSCs differentiate into contractile
myotubes.
Gussoni et al. showed that in an immunodeficient mouse model, marrow-derived cells travel to areas of
induced muscle degeneration, where they can experience myogenic differentiation and contribute to the
regeneration of injured muscle fibers.
Injuries of the physis often result in the development of bone bridges among the epiphysis and the
metaphysis, and 25–35% of these injuries lead to shortening or angular defect.
Stapling, epiphysiodesis, or osteotomies can correct issues in older children; however, treatment is more
difficult in younger patients.
Recently, cultured chondrocytes relocated into physeal defects were shown to correct growth arrest in
animal models.
27. MSCs in Tendon and Ligament Repair
Once these tissues are injured, they heal by forming a tissue of poorer quality than the native tissue.
Multiple biological grafts have been used to reconstruct tendons and ligaments, for example, autografts,
allografts, and biomaterials, but have been plagued by issues with donor site morbidity, shortage, tissue
rejection, and an increased risk of infectious disease transmission.
BMDMSCs were transplanted to various injured tendon sites, resulting in enhanced tissue repair.
BMDMSCs in a fibrin glue vehicle were injected into patellar tendon defects, resulting in enhanced
mature tissue formation and more ordered cell structure than controls.
Healing of the tendon graft-bone interface is critical to the success of ACL surgical reconstruction.
Besides the restoration of the bone-tendon interface, these enhanced grafts have better biomechanical
properties.
Amnion membrane wrapped around ACL.
28. MSCs and Nerve Regeneration
Recovery from peripheral nerve injury is generally poor because of the low regeneration rate of axons,
and intra and extraneural scar tissue development.
MSCs are likely to augment the quantity of Schwann cells and extend their ability to maintain nerve
regeneration.
The inclusion of MSCs, growth factors, and extracellular matrix to conduits and allografts may improve
their performance by creating a favorable environment for axonal regeneration.
In addition, MSCs can be transplanted into denervated muscles, avoiding the predictable sequelae of
denervation, by the prevention of atrophy, and by leaving the tissue more reinnervation-receptive over
extended periods.
Amniotic membrane wrapped around the nerve repair site
29. MSCs in Rotator Cuff Repair
Between 30% and 94% of rotator cuff repairs result in failure, perhaps because the
highly specialized fibrocartilaginous transition area connecting the rotator cuff and the
bone fails to regenerate following repair. The tissue that is formed after the surgery is
a fibrovascular scar tissue, and its mechanical properties are relatively poor [95].
Kida et al. performed drilling to the greater tuberosity to stimulate the liberation of
bone marrow and permit the cells to travel into the suture zone. At 4 and 8 weeks,
MSC migration improved maximum load to failure.
In the study by Gomes, the authors repaired complete rotator cuff tears in 14 patients
using a transosseous approach, enhancing the suture with MSCs obtained from a bone
marrow aspirate from the iliac crest. Based on clinical and magnetic resonance
imaging assessment, all 12 tears healed 12 months after the procedure.
30. Regulatory Aspects on the Use of Stem Cells
Safety and efficacy are the main aspects to be proven derived from well-
controlled clinical trials.
The breakthrough regarding legislation in the United States was the 21st Century
Cures Act of 2016 (Public Law 114-255) that created the designation of
“Regenerative Advanced Therapy” in order to expedite their regulatory
processing.
The FDA only has approved stem cell treatments derived from Bone Marrow
Aspirate Concentrate (BMAC).
31. Hurdles
First, the characterization, differentiation, and expansion procedures used
to prepare these cells must be standardized for homogeneous results.
Second, tissues have a three-dimensional structure, which demands
further research to identify the ideal scaffold for each application based
on the tissue’s structural, biochemical, and biomechanical properties.
Third, neotissues should be functional to replace other injured or
diseased tissues.
Fourth, these neotissues should be immunologically compatible and not
prone to malignancy over time.
Last, treatments using MSCs must be cost effective and widely available
to everyone who needs them.
32. Conclusions
The application of MSCs in medicine is relatively new, but exciting possibilities for the use
of these cells in various diseases.
BMSCs are easy to obtain, isolated and amplified, which provide a wide application prospect
for the treatment of orthopedic diseases.
BMSCs provide a biological, one time, safe and long lasting solution to various orthopaedic
ailments.
Further research is required to refine and reinforce the use of BMCs in orthopaedics.
MSCs exert anti-inflammatory and immunomodulatory effects.
These cells can also be acquired during a surgical procedure and can be cryopreserved for
future use.
