I have received questions in regards to the mechanism of wound healing, and how stem cells play a part. This entry is (hopefully) an easy-to-understand lesson in the biology behind the process. Please feel free to pose any other questions to me and I will do my best to answer!
I will focus on the healing process of the skin, or the dermis. In general, there are four overlapping phases to dermal wound healing: 1. coagulation/hemostasis 2. inflammatory response 3. cell proliferation 4. remodeling.
Hemostasis starts once the initial injury occurs, compromising the integrity of the underlying blood vessels. As blood escapes from these vessels, platelets interact with collagen and other extracellular matrix substances. It is these stimulated platelets that begin the clotting cascade and release clotting factors and inflammatory cytokines.
The inflammatory phase starts within hours of the initial insult, mainly as neutrophils enter the fibrin clot in response to the cytokines. They are followed by leukocytes and macrophages which work together to neutralize foreign substances and help to sterilize the wounded tissue. It is also during this phase that stem cells are activated by pro-inflammatory mediators. Stem cells modulate the immune response and can help inhibit the activity of mast cells and natural killer cells. In this way, stem cells can attenuate the acute immune response. The inflammatory environment also stimulates stem cells to upregulate prostaglandin E2, favoring dermal tissue regeneration. In summary, stem cells favor wound healing over inflammation, while helping to promote functional regeneration during the next phase.
The proliferation phase begins as these immune cells recruit local reparative cells (including more stem cells) from the surrounding area to form what is called granulation tissue. This granulation tissue is well-vascularized, meaning it has a substantial blood supply, and acts as a scaffold for tissue regeneration. Eventually this is what allows for the wound bed to heal and close off. Stem cells help encourage the construction of a viable vascular supply, through the release of many growth factors.
The process concludes with the remodeling phase, wherein cells called fibroblasts help reorganize the extracellular matrix to reinforce the early granulation tissue and also to produce proteins that help regenerate the skin tissue. Stem cells express certain factors that help in this phase to promote growth of healthy and functioning dermal tissue, tissue that resembles uninjured tissue as opposed to scar tissue.
By understanding this entire process and the role stem cells play in it, we can then use a patient's own stem cells to help in the acute healing process. Stem cells directly applied to or injected into a wound can help with healing through multiple mechanisms, adding to their own injury response. There is also accumulating evidence that stem cells given systemically through an IV will find the injured area through complex signaling and still impart a regenerative effect.
I hope this explanation is not too scientific and not overly filled with "medicalese". If so, please let me know....
Wednesday, August 6, 2014
Tuesday, May 27, 2014
The Future of Medicine
The future is looking brighter and brighter when it comes to stem cells and medicine. Stem cells will eventually play a pivotal role in saving lives and curing diseases, along with treating injuries and regenerating our bodies. We are in the early stages of both research and functional use in medical practice.
One of the most intriguing and exciting realms is in bioprinting, or the production of 3-D tissue/organs. Bioprinting involves growing a patient's stem cells in a growth medium in order to have them multiply, then using them to form a "bioink" made out of cell aggregates. This bioink is next placed into cartridges that are essentially syringes with long extension nozzles for printing. Specific software then drives the bioprinter to deposit the bioink cell aggregates into very precise layers. The layers are stacked one upon another and interspersed with hydrogel, a water-based substance that is used as a temporary mold to hold the structure together. The printed tissue is then allowed to grow, and as it matures the hydrogel is removed (usually within the first 24 hours so that this material does not interact with the cells). The finished tissue product can then be used in medical research or as an actual transplanted material for the patient.
Bioprinting is different from traditional tissue engineering that involves culturing cells and subsequently seeding them onto molds or scaffolds. In this more standard model, the mold is designed to look like the intended organ or tissue and is biodegradable. Once the cells mature and produce their own matrix the mold is then removed. It is the timing of the scaffold elimination that is very critical. If removed too quickly the tissue structure can fail. If it degrades too late in the process the tissue may grow into it in a manner that inhibits proper tissue growth and can lead to scarring in patients.
This technology is still in its infancy, as the ability to print tissues has been limited to more basic tissue types. Flat structures such as skin, cartilage, and muscle have been successfully engineered, while tubular structures (blood vessels, trachea, etc.) are a bit more difficult to create. Hollow, non-tubular organs are the next most difficult, such as bladder, stomach, or uterus. And finally, the more solid organs are the trickiest, including the heart, liver and kidneys. These more complex organs involve more cell types, more intricate layering of these cell types, and extensive vascular structure as well.
It is these solid organs that are truly the next frontier in the world of bioprinting. There is some debate as to whether or not we will ever be able to truly create an entire heart or kidney due to the complexity of such organs, and thus the difficulty in matching the minute details as they exist in a functioning human body. We might be able to build a similar organ that functions in the same manner but has differences, most notably in being a simpler design. Another possibility is printing smaller tissue patches that could then be used to repair or augment the body's damaged or diseased organs. An example is engineering a cardiac muscle patch that could then be transplanted to replace an area of heart muscle damaged after a myocardial infarction, or heart attack.
There will no doubt be a time when bioprinted organs take away the need for current transplant surgeries that involve risk to both the recipient and the donor, as well as the potential for rejection and need for immunosuppression.
One of the most intriguing and exciting realms is in bioprinting, or the production of 3-D tissue/organs. Bioprinting involves growing a patient's stem cells in a growth medium in order to have them multiply, then using them to form a "bioink" made out of cell aggregates. This bioink is next placed into cartridges that are essentially syringes with long extension nozzles for printing. Specific software then drives the bioprinter to deposit the bioink cell aggregates into very precise layers. The layers are stacked one upon another and interspersed with hydrogel, a water-based substance that is used as a temporary mold to hold the structure together. The printed tissue is then allowed to grow, and as it matures the hydrogel is removed (usually within the first 24 hours so that this material does not interact with the cells). The finished tissue product can then be used in medical research or as an actual transplanted material for the patient.
Bioprinting is different from traditional tissue engineering that involves culturing cells and subsequently seeding them onto molds or scaffolds. In this more standard model, the mold is designed to look like the intended organ or tissue and is biodegradable. Once the cells mature and produce their own matrix the mold is then removed. It is the timing of the scaffold elimination that is very critical. If removed too quickly the tissue structure can fail. If it degrades too late in the process the tissue may grow into it in a manner that inhibits proper tissue growth and can lead to scarring in patients.
This technology is still in its infancy, as the ability to print tissues has been limited to more basic tissue types. Flat structures such as skin, cartilage, and muscle have been successfully engineered, while tubular structures (blood vessels, trachea, etc.) are a bit more difficult to create. Hollow, non-tubular organs are the next most difficult, such as bladder, stomach, or uterus. And finally, the more solid organs are the trickiest, including the heart, liver and kidneys. These more complex organs involve more cell types, more intricate layering of these cell types, and extensive vascular structure as well.
It is these solid organs that are truly the next frontier in the world of bioprinting. There is some debate as to whether or not we will ever be able to truly create an entire heart or kidney due to the complexity of such organs, and thus the difficulty in matching the minute details as they exist in a functioning human body. We might be able to build a similar organ that functions in the same manner but has differences, most notably in being a simpler design. Another possibility is printing smaller tissue patches that could then be used to repair or augment the body's damaged or diseased organs. An example is engineering a cardiac muscle patch that could then be transplanted to replace an area of heart muscle damaged after a myocardial infarction, or heart attack.
There will no doubt be a time when bioprinted organs take away the need for current transplant surgeries that involve risk to both the recipient and the donor, as well as the potential for rejection and need for immunosuppression.
Tuesday, April 22, 2014
Skin Rejuvenation
Platelet-rich plasma (PRP), as I have discussed in previous posts, has the ability to promote healing and rejuvenation of various tissues. The growth factors that are secreted have shown promise in the production of collagen and other matrix components through the activation of fibroblasts (the most common type of cell in human connective tissue, actually an activated stem cell) in the skin.
An article from the Journal of Drugs in Dermatology, 2010 May; 9(5):466-72, by Redaelli et. al., involved a three month study with 23 patients who received injections of PRP in the face and neck in an effort to promote skin rejuvenation. The patients each received three treatments over the course of the study, with documentation and imaging before and after each session with a final follow-up one month after the last treatment. The injections were given at specific points in both the face and neck, identical in all patients and with every treatment. The study used the following imaging techniques: dermascope, digital camera, and a comprehensive state-of-the-art imaging system with dedicated medical imaging software. The results were evaluated by a special "spider improvement score", a patient's satisfaction score, a doctor's satisfaction score, and a photograph score. In addition, a definitive graduated score was calculated for each patient. Overall the results were satisfactory and showed promise. Also, there were no serious or persistent side-effects. The authors felt like this was a useful therapy for skin rejuvenation.
As a later follow-up to this, a group of researchers (Kim, et.al.) in Korea published an article in the Annals of Dermatology, 2011 Nov.; 23(4): 424-31, evaluating the actual effects of PRP on dermal fibroblasts. This study looked at the effects of PRP on matrix protein synthesis, collagen production, and fibroblast cell proliferation. PRP showed an increase in the expression of type 1 collagen, MMP-1 protein (matrix metalloproteinase), and mRNA in human dermal fibroblasts, thus verifying that PRP does promote tissue remodeling. The researchers hypothesized that the PRP may promote extracellular matrix remodeling through the removal of photo-damaged components and through the induction of new collagen synthesis by the fibroblasts, which in turn proliferate by their stimulation.
It is felt that PRP can be beneficial as either a stand-alone procedure, or as an adjuvant therapy with lasers for skin rejuvenation. We are currently interested in comparing the results of both a superficial application and injections, or possibly even a combination of both. In addition, the use of stem cells might improve the results even more, although at a higher dollar cost.
An article from the Journal of Drugs in Dermatology, 2010 May; 9(5):466-72, by Redaelli et. al., involved a three month study with 23 patients who received injections of PRP in the face and neck in an effort to promote skin rejuvenation. The patients each received three treatments over the course of the study, with documentation and imaging before and after each session with a final follow-up one month after the last treatment. The injections were given at specific points in both the face and neck, identical in all patients and with every treatment. The study used the following imaging techniques: dermascope, digital camera, and a comprehensive state-of-the-art imaging system with dedicated medical imaging software. The results were evaluated by a special "spider improvement score", a patient's satisfaction score, a doctor's satisfaction score, and a photograph score. In addition, a definitive graduated score was calculated for each patient. Overall the results were satisfactory and showed promise. Also, there were no serious or persistent side-effects. The authors felt like this was a useful therapy for skin rejuvenation.
As a later follow-up to this, a group of researchers (Kim, et.al.) in Korea published an article in the Annals of Dermatology, 2011 Nov.; 23(4): 424-31, evaluating the actual effects of PRP on dermal fibroblasts. This study looked at the effects of PRP on matrix protein synthesis, collagen production, and fibroblast cell proliferation. PRP showed an increase in the expression of type 1 collagen, MMP-1 protein (matrix metalloproteinase), and mRNA in human dermal fibroblasts, thus verifying that PRP does promote tissue remodeling. The researchers hypothesized that the PRP may promote extracellular matrix remodeling through the removal of photo-damaged components and through the induction of new collagen synthesis by the fibroblasts, which in turn proliferate by their stimulation.
It is felt that PRP can be beneficial as either a stand-alone procedure, or as an adjuvant therapy with lasers for skin rejuvenation. We are currently interested in comparing the results of both a superficial application and injections, or possibly even a combination of both. In addition, the use of stem cells might improve the results even more, although at a higher dollar cost.
Tuesday, January 28, 2014
Meniscus Knee Surgery and Stem Cell Therapy
The latest in orthopedic surgery and stem cell therapy was discussed in a study that appeared in the January 2014 issue of the Journal of Bone and Joint Surgery (Volume 96, Issue 2). The study, whose full title is "Adult Human Mesenchymal Stem Cells (MSC) Delivered via Intra-Articular Injection to the Knee, Following Partial Medial Meniscectomy", followed groups of patients who received a single injection of stem cells after knee surgery.
The study was designed with three groups of patients: those receiving a "low-dose" of 50 million stem cells within 7-10 days after surgery, those receiving a higher dose of 100 million stem cells, and a control group receiving sodium hyaluronate without stem cells. The surgery performed is called a partial meniscectomy, a procedure that is used to treat tears in the meniscus by removing all or part of the torn cartilage. There were a total of 55 patients in the study.
The key findings of this study were as follows:
1. There was no abnormal/ectopic tissue formation.
2. There were no "clinically important" safety issues identified.
3. There was "significantly increased meniscal volume" by MRI in 24% of the "low-dose" patients, and 6% in the higher dosed patients at one year. There was no statistical increase in either group at 2 years. A "significant increase" was defined as at least a 15% increase.
4. There was no statistical increase in meniscal volume in any of the control group patients.
5. Stem cell patients with osteoarthritis showed a reduction in pain; control group patients experienced no decrease in pain.
These findings are consistent with what has been found in the literature and with the results we have experienced in our practice. The stem cells show improvement both objectively (increased meniscal volume by MRI) and subjectively (decreased pain). It is interesting that this study was performed on post-surgical patients, as the stem cell therapy might have eliminated the need for surgery if performed alone. However, it does show that stem cells can be beneficial for those patients who are determined to have surgery.
The study was designed with three groups of patients: those receiving a "low-dose" of 50 million stem cells within 7-10 days after surgery, those receiving a higher dose of 100 million stem cells, and a control group receiving sodium hyaluronate without stem cells. The surgery performed is called a partial meniscectomy, a procedure that is used to treat tears in the meniscus by removing all or part of the torn cartilage. There were a total of 55 patients in the study.
The key findings of this study were as follows:
1. There was no abnormal/ectopic tissue formation.
2. There were no "clinically important" safety issues identified.
3. There was "significantly increased meniscal volume" by MRI in 24% of the "low-dose" patients, and 6% in the higher dosed patients at one year. There was no statistical increase in either group at 2 years. A "significant increase" was defined as at least a 15% increase.
4. There was no statistical increase in meniscal volume in any of the control group patients.
5. Stem cell patients with osteoarthritis showed a reduction in pain; control group patients experienced no decrease in pain.
These findings are consistent with what has been found in the literature and with the results we have experienced in our practice. The stem cells show improvement both objectively (increased meniscal volume by MRI) and subjectively (decreased pain). It is interesting that this study was performed on post-surgical patients, as the stem cell therapy might have eliminated the need for surgery if performed alone. However, it does show that stem cells can be beneficial for those patients who are determined to have surgery.
Friday, December 13, 2013
Stem Cells and Inflammation
This entry is in regards to an interesting review article in the October, 2013 Stem Cells journal, by Darwin J. Prockop, M.D., Ph.D from Texas A&M University. He writes about two specific negative feedback loops that stem cells introduce into generic pathways of inflammation.
(I apologize about the technical aspect of this blog entry, but I found it difficult to put a lot of the medical terminology into layperson terms.)
The first feedback loop is brought about by pro-inflammatory mediators from certain sensor cells that activate stem cells and upregulate the expression of COX2 and other parts of the arachidonic acid pathway. This then causes the stem cells to secrete more prostaglandin E2 (PGE2) which then appears to cause pro-inflammatory macrophages to instead secrete anti-inflammatory mediators (including interleukin-10 and interleukin-1).
The second negative feedback loop also involves stem cell activation from mediators. This activation causes stem cells to increase the expression of certain genes, specifically the anti-inflammatory protein TNF-alpha stimulated gene/protein 6 (TSG-6). This results in a decrease in TNF-alpha and other mediators that stimulate inflammation.
These two loops provide evidence that stem cells have a definite effect in decreasing inflammation, a finding that once again shows promise for stem cells as a treatment modality.
The lab testing evidence for these two loops was developed primarily in experiments wherein the stem cells were given after an acute tissue injury that induced the inflammatory response. The author notes that further investigation is needed to determine if these feedback loops exist in situations such as mild inflammation or unresolved inflammation due to a chronic disease process. It is also intriguing to note if these negative feedback loops would exist in all patients, or if genetic differences would influence them.
These negative feedback loops are only one portion of the role that stem cells have in the healing response. There are many other factors involved, including stimulation of growth factors, increased vascularization, recruitment of more host stem cells, modulation of the immune response, reduction of reactive oxygen species, transdifferentiation of cell types, and increased paracrine signaling, among many other methods.
(I apologize about the technical aspect of this blog entry, but I found it difficult to put a lot of the medical terminology into layperson terms.)
The first feedback loop is brought about by pro-inflammatory mediators from certain sensor cells that activate stem cells and upregulate the expression of COX2 and other parts of the arachidonic acid pathway. This then causes the stem cells to secrete more prostaglandin E2 (PGE2) which then appears to cause pro-inflammatory macrophages to instead secrete anti-inflammatory mediators (including interleukin-10 and interleukin-1).
The second negative feedback loop also involves stem cell activation from mediators. This activation causes stem cells to increase the expression of certain genes, specifically the anti-inflammatory protein TNF-alpha stimulated gene/protein 6 (TSG-6). This results in a decrease in TNF-alpha and other mediators that stimulate inflammation.
These two loops provide evidence that stem cells have a definite effect in decreasing inflammation, a finding that once again shows promise for stem cells as a treatment modality.
The lab testing evidence for these two loops was developed primarily in experiments wherein the stem cells were given after an acute tissue injury that induced the inflammatory response. The author notes that further investigation is needed to determine if these feedback loops exist in situations such as mild inflammation or unresolved inflammation due to a chronic disease process. It is also intriguing to note if these negative feedback loops would exist in all patients, or if genetic differences would influence them.
These negative feedback loops are only one portion of the role that stem cells have in the healing response. There are many other factors involved, including stimulation of growth factors, increased vascularization, recruitment of more host stem cells, modulation of the immune response, reduction of reactive oxygen species, transdifferentiation of cell types, and increased paracrine signaling, among many other methods.
Tuesday, October 1, 2013
Stem Cell Mechanism of Action
A recent study from Laura Shin and Daniel Peterson in Stem Cells Translational Medicine (2013; 2:33-42) attempts to clarify exactly how stem cells given as a treatment actually induce healing. The conventional theory has been that these cells act by differentiating once inside the body, as in they change into the cell types that the body needs to repair and heal itself. This equates to the analogy that they act as seeds that then grow into the new tissue.
However, this recent study seems to point to a different mechanism of action.The researchers used human mesenchymal stem cells in mice that were given excisional wounds that were splinted open. The splint wound model more closely duplicates how human wounds heal by granulation and re-epithelialization (rather than by skin contraction, the normal repair mechanism in rodents). The treated mice were given a pair of these wounds, one that received an engraftment of stem cells and the other left untreated. The stem cells were genetically modified so the researchers could trace the cells after giving them to the mice. There was also a control group who had similar paired wounds, but received no stem cells.
The results of this study showed that the animals that received stem cell therapy had accelerated healing when compared to the control group. However, the stem cells themselves did not seem to be the cells directly involved in the wound repair. The traced stem cells were abundantly evident in the treated wound just one day after delivery, but the numbers were dramatically reduced by day 5, and these cells were not even detectable in the wound bed by day 10.
The wounds that received no stem cells showed a rather significant delay in closure compared to those receiving stem cells. The stem cells were also only engrafted into one of the paired wounds on each treated mouse, but the non-treated wound on that mouse showed a moderate improvement in healing over the non-stem cell mice as well.
The results seem to indicate that the improved closure of the wounds despite the relatively rapid decrease in engrafted stem cell numbers may be due to signalling within the wound bed that helps to modulate and direct the host's own cells. The injected stem cells appear to recruit the patient's own stem cells to the area of injury, both at the engrafted site as well as distant sites in the body to some degree. These signals also seem to be maintained after the engrafted stem cells are gone. Thus the authors propose that while the stem cells are useful for healing, it is the subsequent healing response that they induce that ultimately leads to wound repair. If this is truly the manner that stem cells are involved in the healing process, it is the signals they produce that are the key to success with future therapies.
However, this recent study seems to point to a different mechanism of action.The researchers used human mesenchymal stem cells in mice that were given excisional wounds that were splinted open. The splint wound model more closely duplicates how human wounds heal by granulation and re-epithelialization (rather than by skin contraction, the normal repair mechanism in rodents). The treated mice were given a pair of these wounds, one that received an engraftment of stem cells and the other left untreated. The stem cells were genetically modified so the researchers could trace the cells after giving them to the mice. There was also a control group who had similar paired wounds, but received no stem cells.
The results of this study showed that the animals that received stem cell therapy had accelerated healing when compared to the control group. However, the stem cells themselves did not seem to be the cells directly involved in the wound repair. The traced stem cells were abundantly evident in the treated wound just one day after delivery, but the numbers were dramatically reduced by day 5, and these cells were not even detectable in the wound bed by day 10.
The wounds that received no stem cells showed a rather significant delay in closure compared to those receiving stem cells. The stem cells were also only engrafted into one of the paired wounds on each treated mouse, but the non-treated wound on that mouse showed a moderate improvement in healing over the non-stem cell mice as well.
The results seem to indicate that the improved closure of the wounds despite the relatively rapid decrease in engrafted stem cell numbers may be due to signalling within the wound bed that helps to modulate and direct the host's own cells. The injected stem cells appear to recruit the patient's own stem cells to the area of injury, both at the engrafted site as well as distant sites in the body to some degree. These signals also seem to be maintained after the engrafted stem cells are gone. Thus the authors propose that while the stem cells are useful for healing, it is the subsequent healing response that they induce that ultimately leads to wound repair. If this is truly the manner that stem cells are involved in the healing process, it is the signals they produce that are the key to success with future therapies.
Thursday, August 1, 2013
Stem Cells From Fat Versus Bone Marrow
A recent study published in the journal "Stem Cells Translational Medicine" seems to indicate that adipose-derived stem cells are more potent than bone marrow derived cells when comparing how they each modulate the immune system.
Functionally, both cell types responded similarly. They each had the capacity to differentiate toward both osteogenic and adipogenic lineages. In terms of the immune system response, both had similar surface marker expression. However, the adipose-derived stem cells showed a significantly higher level of suppression of peripheral blood mononuclear cell proliferation when used in equal numbers. Approximately three times the number of bone marrow derived stem cells were necessary to get the same suppressive effect.
This effect appears to be related to a decrease in inflammatory cytokines and an increase in certain anti-inflammatory cytokines. Cytokines are immunomodulating agents, such as interleukins and interferons.
This marks a significant finding in using stem cells for therapeutic applications. The frequency of stem cells in bone marrow is comparatively low, with stem cells accounting for only 0.001% - 0.01% of the total mononuclear cell fraction. Also, bone marrow aspiration is relatively invasive. Adipose tissue, however, contains approximately 500-fold greater frequency of stem cells, and collection via lipoaspiration is simple and easily tolerated.
The study also showed that adipose cells secrete higher levels of a multitude of cytokines that have been implicated in how stem cells modulate our immune systems.
Because of these many factors, adipose-derived stem cells appear to be a much better choice for cell-based therapies.
-from Stem Cells Translational Medicine 2013;2:455-463
Functionally, both cell types responded similarly. They each had the capacity to differentiate toward both osteogenic and adipogenic lineages. In terms of the immune system response, both had similar surface marker expression. However, the adipose-derived stem cells showed a significantly higher level of suppression of peripheral blood mononuclear cell proliferation when used in equal numbers. Approximately three times the number of bone marrow derived stem cells were necessary to get the same suppressive effect.
This effect appears to be related to a decrease in inflammatory cytokines and an increase in certain anti-inflammatory cytokines. Cytokines are immunomodulating agents, such as interleukins and interferons.
This marks a significant finding in using stem cells for therapeutic applications. The frequency of stem cells in bone marrow is comparatively low, with stem cells accounting for only 0.001% - 0.01% of the total mononuclear cell fraction. Also, bone marrow aspiration is relatively invasive. Adipose tissue, however, contains approximately 500-fold greater frequency of stem cells, and collection via lipoaspiration is simple and easily tolerated.
The study also showed that adipose cells secrete higher levels of a multitude of cytokines that have been implicated in how stem cells modulate our immune systems.
Because of these many factors, adipose-derived stem cells appear to be a much better choice for cell-based therapies.
-from Stem Cells Translational Medicine 2013;2:455-463
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