A Comparative study of the Anticonvulsant effect of Nimodipine andKetamine combination with standardanticonvulsant drug in Rodents Prasanand S1, Pushpalatha C2, Mohsin MD3, Sam Pavan Kumar G4, Gundappa Rao S5 Aim of the study: To evaluate and compare the anticonvulsant property of nimodipine andketamine combination with a standard drug like Sodium valproate in electrically and chemically
Regenerativehealth.co.zaPain Physician 2008; 11:3:343-353 • ISSN 1533-3159
Increased Knee Cartilage Volume in
Degenerative Joint Disease using
Percutaneously Implanted, Autologous
Mesenchymal Stem Cells
Christopher J. Centeno, MD1,2, Dan Busse MD1, John Kisiday, PhD3, Cristin Keohan1,2, Michael Freeman, PhD4, and David Karli, MD5 From: 1Regenerative Sciences Inc (RSI), 2Centeno-Schultz Clinic; 3Colorado State Background: The ability to repair tissue via percutaneous means may allow
University, Fort Collins, CO; 4Oregon interventional pain physicians to manage a wide variety of diseases including Health Sciences University and School of Medicine; and the 5Steadman Hawkins peripheral joint injuries and osteoarthritis. This review will highlight the devel- Clinic, Vail, CO.
opments in cellular medicine that may soon permit interventional pain manage-ment physicians to treat a much wider variety of clinical conditions and highlight Dr. Centeno is with Regenerative Sciences an interventional case study using these technologies Inc (RSI), Centeno-Schultz Clinic, Westminster, CO. Objective: To determine if isolated and expanded human autologous mes-
Dr. Busse is with the Centeno-Schultz Clinic, Westminister, CO.
enchymal stem cells could effectively regenerate cartilage and meniscal tissue Dr. Kisiday is with Colorado State when percutaneously injected into knees. University, Fort Collins, CO.
Ms. Keohan is with the Centeno-Schultz Design: Case Study
Clinic and Regenerative Sciences Inc.
Dr. Freeman is with the Oregon Health Setting: Private Interventional Pain Management practice.
Sciences University and School of Medicine, Salem, OR.
Dr. Karli is with Steadman Hawkins Clinic, Methods: An IRB approved study with a consenting volunteer in which mes-
enchymal stem cells were isolated and cultured ex-vivo from bone marrow aspi-ration of the iliac crest. The mesenchymal stem cells were then percutaneously Address correspondence: injected into the subject's knee with MRI proven degenerative joint disease. Pre- and post-treatment subjective visual analog pain scores, physical therapy assess- Centeno-Schultz Clinic 11080 Circle Point Rd ments, and MRIs measured clinical and radiographic changes. Buliding 2, Suite 140 Westminster, CO 80020 Results: At 24 weeks post-injection, the patient had statistically significant car-
tilage and meniscus growth on MRI, as well as increased range of motion and decreased modified VAS pain scores. Disclaimer: Dr. Centeno, Dr. Busse and Ms. Koen have financial stake in Regenerative Sciences Inc. which is involved in stem cell Conclusion: The described process of autologous mesenchymal stem cell cul-
research and treatment.
ture and percutaneous injection into a knee with symptomatic and radiograph- Conflict of interest: None.
ic degenerative joint disease resulted in significant cartilage growth, decreased pain and increased joint mobility in this patient. This has significant future impli- Manuscript received: 12/27/2007 cations for minimally invasive treatment of osteoarthritis and meniscal injury. Revised manuscript received: 03/05/2008 Accepted for publication: 04/17/2008 Key words: autologous mesenchymal stem cells, osteoarthritis, knee, carti-
Free full manuscript: Pain Physician 2008; 11:3:343-353
Pain Physician: May/June 2008:11:343-353 let Rich Plasma (PRP) which contains a high concentra-tion of stored autologous growth factors. Red blood Interventional pain practices in the U.S. have helped
to bridge the gap between surgical solutions and conservative care such as rehabilitation. However, cells are separated from PRP which typically contains to date the main focus of interventional pain medicine a hematocrit below 5%. Most commercially available has been spinal. Regenerative medicine has been PRP centrifuge products have the ability to produce a loosely defined as the ability to regenerate tissue 4 – 7 fold increase in platelet concentration compared rather than surgically extracting or altering that tissue. The ability to repair tissue via percutaneous means By exposing PRP to thrombin, platelet degranula- may allow interventional pain physicians to manage tion is induced and a concentrated pool of autologous a wide variety of diseases including peripheral joint growth factors in physiologic combinations can be injuries and osteoarthritis. This review will highlight delivered to injured tissue as a therapeutic modality the developments in cellular medicine that may soon to augment natural regenerative pathways. Once ac- permit interventional pain management physicians tivated by thrombin, PRP is termed Platelet Gel (PG), to treat a much wider variety of clinical conditions due to initiation of clotting pathways mediated by fi- and also highlights an interventional case study using brin in the serum.
Initial PRP studies used bovine thrombin as an Regenerative medicine can be broken into 3 main activator. It has since been discovered that bovine areas: platelet augmentation, stem cell isolates, and thrombin can lead to the development of antibod- recombinant growth factor amplification. Frequently ies to clotting factors V, XI, and autologous thrombin. these approaches have been combined. For example, In rare case reports, this has lead to systemic and life adult stem cells require various growth factors to threatening coagulaopathies with multisystem failure maintain their growth and engraftment (1-5). As a re- (8). New techniques have since been developed to sult, stem cells are usually transplanted with growth generate autologous thrombin from the same blood sample used to make PRP, with superior properties to The platelet augmentation approach is based on bovine thrombin. the concept that platelets contain key growth factors Percutaneously, PRP can be implanted into target such as Platelet Derived Growth Factors (PDGFs), Trans- tissue along with autologous thrombin via a double forming Growth Factors (TGFs), Fibroblast Growth Fac- lumen injection technique. Alternatively, PRP can be tors (FGFs), and various forms of Interleukins (IL) (6). activated by thrombin in vitro to produce a lower vis-cocity Platelet Lysate (PL) containing growth factors Autologous PlAtelet APProAches
without platelet tissue or a fibrin matrix. PL has been The platelet augmentation approach is based on successfully used as a cell culture additive to facilitate the concept that platelets, via an intricate vesicular growth and differentiation of autologous mesenchy- storage system, contain critical growth factors and me- mal stem cells (3,9-11).
diators of tissue repair pathways. In response to tissue Sound basic science and animal testing data, injury, a complex cascade of cellular and noncellular along with a handful of human clinical trials have signals triggers platelet receptors, resulting in expul- demonstrated a strong safety profile and encouraging sion of these growth factors within the site of injury. therapeutic results. Mishra and Pavelko (12) have in- This process is known as degranulation and initiates vestigated PRP injections into chronic elbow tendon- cellular proliferation and a tissue repair response. One osis, reporting a greater than 90% reduction in pain of the key triggers for platelet activation is the protein up to 2 years out from the intervention. Aspenberg thrombin, which has been shown to induce immediate and Virchenko (13) found that a single PRP injection platelet growth factor release in vitro in a dose depen- into a transected rat Achilles tendon increased tis- dant fashion (7). sue strength by 30%, with histology showing greater Understanding platelet physiology has led to the maturation of tendon cells vs. control. Based on work concept of utilizing platelet growth factors to develop by Anitua et al (8), the application of PG therapy has novel and natural regenerative therapies. Several com- broad applications within orthopedic medicine. They mercially available centrifugation systems can isolate hypothesize that the released growth factors have a and concentrate platelets from an autologous blood chemotactic and mitogenic effect on mesenchymal sample. The resulting blood isolate is known as Plate- stem cells (MSCs) and osteoblasts when applied to Increased Knee Cartilage Volume Using Autologous Mesenchymal Stem Cells bony tissues. These authors have also hypothesized fibrocartilage repair. In addition, this cell population other mechanisms of action in addition to recruitment has also been shown to assist in the repair of non- of progenitor cells to replace damaged tissue, includ- union fractures (31). For this application, bed-side cen- ing angiogenesis, local anabolic stimulation of cellu- trifugation is commonly used. Again, these techniques lar protein synthesis, and gene expression, leading to produce a very dilute MSC population, usually a yield proliferation, tissue remodeling, and production of of 1 in 10,000 – 1,000,000 of the nucleated cells (32). new extracellular matrix (ECM). Additionally, isolated bone marrow nucleated cells implanted into degenerated human peripheral joints tem cells And regenerAtive medicine
have shown some promise for joint repair (33). Our Embryonic stem cells have been in the popular own research in this area showed some capability of media for many years and have shown promise sci- regenerating bony cortex over a hip subchondral cyst, entifically while creating significant controversy. As a but cartilage percutaneous replacement seemed be- result, many researchers have focused on adult stem yond the capability of this cell population.
cells, or stem cells isolated from adult humans that Since it is MSCs that are capable of differentiat- can be transplanted into damaged tissue. Mesenchy- ing into cartilage, their use as cellular building blocks mal stem cells (MSC) are pluripotent cells found in that can be implanted via fluoroscopically guided multiple human adult tissues including bone marrow, percutaneous procedures has some face validity. As synovial tissues, and adipose tissues. Since they are de- discussed above, the number of MSCs that can be rived from the mesoderm, they have been shown to isolated from bone marrow is fairly limited. As a re- differentiate into bone, cartilage, muscle, and adipose sult, most research in cartilage regeneration has fo- tissue (14). Because of their multi-potent capabilities, cused on the use of culture expanded cells (34-37). MSC lineages have been used successfully in animal This means that cells are isolated and then placed models to regenerate articular cartilage and in human into various growth factors to be grown to higher models to regenerate bone (15-28). Of importance to numbers over a period of weeks in an ex-vivo mono- the interventional pain management community is layer culture. The most common method of isolating recent research demonstrating that articular cartilage the very scarce MSCs from the more numerous bone may be able to be repaired via percutaneous introduc- marrow nucleated cells is through adhesion to plas- tion of MSCs (28-30). tic in tissue culture (38). MSCs will adhere to plastic, The use of autologous adult bone marrow derived whereas the remainder of the nucleated cell popula- stem cells in research can again be segregated into 3 tion will not adhere. As a result, the adherent colo- main areas: nucleated cell isolates, isolated MSCs with- nies are collected and then over several steps further out culture expansion, and isolated MSCs with culture "filtered" out of the larger nucleated cell population expansion. Most of the adult bone marrow consists of using adhesion. Once a pure MSC population is iso- blood cells in various stages of differentiation. These lated, the tissue culture is transferred to monolayer, marrow components can be divided into plasma, red where the cells can continue to grow adherent to the blood cells, platelets, and nucleated cells. The adult plastic flask, while they are covered by a thin layer of stem cell fraction is present in the nucleated cells of basal nutrient media and growth factors. As the cells the marrow. Most of these cells are CD34+ heme pro- cover the surfaces of the plastic flasks, they are pe- genitors (destined to differentiate into blood compo- riodically removed from those surfaces with trypsin nents), while very few are actually MSCs capable of and reseeded in a greater number of flasks with new differentiating into bone, cartilage, or muscle. In small media (nutrients and growth factors). This one cycle animal models these CD34+ cells can transdifferenti- of cell collection, seeding, and feeding is known in ate into MSCs, but in primates this does not appear the cell culture vernacular as a "passage." MSCs are to be the case (29). As a result, that leaves the small usually harvested between the third and fifth pas- number of MSCs in the marrow as cells capable of dif- sage (several weeks total culture time) and at that ferentiating into tissues of interest to pain medicine. point have been grown to between 100 – 10,000 Marrow nucleated cells are used every day in re- times more cells than harvested (39). generative orthopedics. The knee micro fracture tech- As discussed above, once cells are ready for reim- nique popularized by Steadman et al (30) relies on the planation, they are usually transferred with growth release of these cells into a cartilage lesion to initiate factors to allow for continued cell growth and engraft- Pain Physician: May/June 2008:11:343-353 ment to the damaged tissue. At some point, a signal is introduced (either in culture or after transplant to The research protocol was approved through a the damaged tissue) for the cells to differentiate into non-profit Institutional Review Board (The Spinal In- the end tissue (in this case cartilage). Various elements jury Foundation, Westminster CO).
of the local microenvironment can affect MSC differ- Inclusion criteria were as follows: entiation (1,18,31,40,41). Several authors have shown 1. Male or female patients, 18 – 65 years of age.
that nano doses of dexamethasone acts as a potent differentiating agent for MSCs toward a chrondrogen- ic lineage (34,42,43). This concept is to be separated 3. Persisting intrusive pain resulting from the condi- from clinical doses of corticosteroids, which have been tion identified in (2). To ensure that the diagnosis shown to cause cellular apoptosis and joint destruc- of intrusive osteoarthritis was accurate, diagnostic tion over time (44-48). blocks were used. Confirmation that a joint was In addition, it's thought that a scaffolding mate- a primary cause of the patients' pain was accom- rial might be needed to allow the MSCs to attach and plished with a fluoroscopically guided injection of engraft (38-41). Many different types of scaffolding 0.75% Marcaine and 4% Lidocaine into the joint material have been used. For example, MSCs can be space followed by complete pain relief. seeded on an allograft such as an ACL ligament (49). 4. The patients had been evaluated by a board certi- In addition, injectable, self-assembling scaffolds are fied orthopedic surgeon and informed that they common such as fibrin glue, hyaluronans, or mixtures were a knee arthroplasty candidate.
of both (50,51). Exclusion criteria: 1. Active inflammatory or connective tissue disease rowth FActors in regenerAtive
(i.e. lupus, fibromyalgia, RA).
2. Active non-corrected endocrine disorder poten- Recombinant human growth factors have just be- tially associated with symptoms (i.e. hypothyroid- gun to be used in modern clinical practice. These are ism, diabetes).
proteins in the cytokine family that have the ability to 3. Active neurologic disorder potentially associated promote growth of certain tissues. The first of these with symptoms (i.e. peripheral neuropathy, mul- to be available in spinal surgery practices is the Bone tiple sclerosis).
Morphogenic Protein (BMP) family. Both BMP-2 and 4. Severe cardiac disease.
BMP-7 are currently being used to promote fusion. 5. Pulmonary disease requiring medication usage.
(52,53). As of yet unpublished animal trials with these 6. A history of dyspnea or other reactions to transfu- growth factors may show some promise in repair of sion of homologous blood products.
peripheral joints (54). In addition, FGF-2 has also re- Pre-procedure data collection: cently been used in unpublished, small animal studies 1. CBC and SMAC to rule out unknown medical con- to promote disc repair. The TGF family has also shown dition (within 3 months of procedure).
promise in the same areas (55). 2. The patient was surveyed for activity level.
The present case study reports on a successful A 46-year-old male was actively recruited by his clinical result using percutaneously implanted, autolo- pain physician from a private interventional pain man- gous culture expanded MSCs with nucleated cells and agement practice based on his diagnosis of OA caus- physiologic doses of dexamethasone as a differentiat- ing significant ongoing pain and disability, and their ing agent. This patient was treated with a completely willingness to proceed with the study. This patient was autologous processes, where mesenchymal stem cells provided with extensive informed consent prior to be- were isolated from the iliac crest via fluoroscopically ing enrolled in the study. guided percutaneous procedure, ex-vivo culture ex- panded in a clinical lab set up for this purpose, and Atient history
then reintroduced via fluoroscopy with autologous The patient presented with a several year history growth factors isolated from platelets and marrow of left greater than right knee pain he attributed to nucleated cells.
multiple injuries from parachute jumping and long Increased Knee Cartilage Volume Using Autologous Mesenchymal Stem Cells marches with heavy weights during his military ser- co's Phosphate Buffered Saline) with 1mM EDTA (eth- vice. The pain is constant, daily, 3-7/10 in intensity, and ylene diamine tetraacetic acid) and the MSCs were associated with "locking up" several times a month. reseeded at a density of 12,000 cells/cm2 in αMEM + He had 3 arthroscopic surgeries, including microfrac- 5%, 10%, or 20% PL. Primary cells derived from the ture, on his right knee with 5 to 7 years of transient bone marrow were designated as passage 0 and each relief. His left knee, however, has undergone no surgi- subsequent reseeding of MSC was considered one fur- cal interventions. He has been told by his orthopae- dists that his only option going forward is total knee After MSCs had been grown to the fifth passage, they were suspended in PBS. The patient returned to Physical exam of the left knee was remarkable for the clinic and was consented in writing. The patient a slight effusion, bilateral joint line tenderness, posi- was then placed prone on a fluoroscopy table and tive McMurray's and Lachman's, and negative tests for the bilateral PSIS area was prepped with betadine ligamentous instability. A pre-treatment 3Tesla MRI and sterile drapes. The skin and deeper tissues were showed degenerative joint disease.
then anesthetized with 1% Lidocaine. A sterile trocar For one week prior to the marrow harvest proce- was then inserted under fluoroscopically guidance to dure the patient was restricted from taking corticoste- the superior medial corner of the iliac wing and 50 roids or NSAIDs. Coincident with the marrow harvest mL of marrow was drawn. This sample was sent to procedure, approximately 200cc of heparinized IV ve- the lab for nucleated cell isolation. Red blood cells nous blood was drawn to be used for PL. To prepare were separated from the majority of nucleated cells PL, PRP was prepared via centrifugation at 200g to in the whole marrow via centrifugation at 200g for separate plasma/platelets from the red blood cells. The supernatant was then drawn off and platelets The patient returned to the OR a few hours lat- pelleted at 1000g centrifugation. The platelets were er and was placed supine with the right knee bent then re-suspended at an approximate concentration at 45 degrees and re-prepped using betadine and of 1 x 109 per mL. This was considered 100% PL, which sterile gloves. A 25-gauge 2-inch needle was then was then diluted to between 10 – 20% as supplement inserted through a medial inferior approach under for serum free cell culture media. C-arm guidance. Once the joint space was attained, The patient was then placed prone on an OR ta- 0.25 mL of Isovue contrast diluted 50% with PBS ble and the area to be harvested was numbed with was injected. Once good medial intraarticular flow 1% Lidocaine, and a sterile disposable trocar was used was established, 22.4 million MSCs suspended in PBS to draw 10 mL of marrow blood from the right Poste- were injected, followed by 1 mL of nucleated cells rior Superior Iliac Spine area and 10 mL from the left suspensed in PBS and 1 mL of 10% PL. The patient was instructed to remain still for one hour to allow Whole marrow was centrifuged at 100g for 4 – 6 for cell attachment and then was instructed to main- minutes to separate the plasma from the RBCs. The tain activity as tolerated. The patient returned for 2 plasma was removed, placed in a separate tube, and additional 10% intraarticular knee PL injections (1 centrifuged at 1000g for 10 minutes to pellet the nu- mL) at week 1 and week 2 (post-transplantation). cleated cell fraction. The nucleated cells were washed With the 2-week post transplant PL supplementa- once in PBS, counted, and then re-suspended in DMEM tion, 1 mL of 10 ng/mL dexamethasone was also (Dulbecco's medium) + 10% PL and seeded at 1 x 106 cells/cm2 in monolayer flask culture. Cultures were Modified VAS questionnaires and Functional incubated at 37°C/5% CO2 in a humidified environ- Rating Index (28,29) questionnaires were provided ment. The culture medium was changed after 3 days, to the patient and administered before the proce- removing the majority of the non-adherent cell popu- dure, one month after the procedure, and 3 months lation. MSC colonies developed 6 – 12 days after seed- after the procedure. In particular, additional VAS ing. After growing to near confluence, the colonies data was calculated by multiplying the modified were trypsinized over 2 – 3 minutes such that only the numerical VAS for knee pain by the frequency of colony-forming MSCs detached. To avoid confluence, that complaint. Range of motion measurements cells were harvested with 1x trypsin in DPBS (Dulbec- of the knee were measured by a physical therapist Pain Physician: May/June 2008:11:343-353 before the procedure, one month post-procedure, and 3 months post-procedure. In addition, pre-pro- Cell yields upon initial marrow harvest and pro- cedure MRIs were obtained on a GE 3.0 T magnet cessing are discussed below in Table 1. Note the dis- with Proton Density Fast Spin sequences in the parity in the nucleated cell yield from left to right sagittal coronal planes. Post-procedure images at sites. Since MSCs represent approximately 1 in 10,000 one month and at 3 months were obtained using nucleated marrow cells, nucleated cell yield is a likely matching excitation times (NEX), repetition times proxy for total MSCs obtained by the marrow draw.
(TR), and echo times (TE). Quantitative meniscus Table 1 shows the cell yields from marrow draw.
and articular cartilage volume analysis was carried Table 2 shows MSC growth per day in mono- out using commercially available image processing layer culture and Table 3 shows growth per passage. software (OSIRIS- Digital Imaging Unit, Division of A passage was defined as a change of tissue culture Medical Informatics, University Hospital of Geneva) using 3 traces by the same examiner of each region Figures 1 and 2 show representative sagittal pro- of interest. Standard deviation from the mean was ton density fast spin MRI images from a GE 3.0T mag- calculated for these 3 traces. The area of the medial net with matching TR and TE from pre-procedure and weight bearing femoral defect was also traced and 6 months post-procedure. Cartilage volume analysis is calculated in a similar manner.
Table 1. Cell yields from marrow draw. Platelet Count From Whole
Marrow Draw Site
Nucleated Cell Count
Left Posterior Superior Iliac Spine Right Posterior Superior Iliac Spine Table 2. MSC growth per day in millions. Table 3. MSC growth in millions per tissue culture passage. Cell Growth
Cell Growth per Passage
Days in colony = 10
Increased Knee Cartilage Volume Using Autologous Mesenchymal Stem Cells Fig.1. Pre-injection. Left shows cartilatge and right shows meniscus. Fig.2. Six months post-injection. Left shows cartilatge and right shows meniscus. Table 4: Cartilage volume analysis in mm3. % Change from
Cartilage surface Cartilage surface Cartilage surface
Pain Physician: May/June 2008:11:343-353 Fig. 3. Intraarticular placement of 25-gauge 2-inch Quinke Fig. 4. Placement of the trocar into the PSIS region for needle through a medial inferior skin site. Dye flow as seen bone marrow aspiration using fluoroscopic guidance. on AP fluoroscopy. Table 5: Patient outcomes. Functional Rating Index and Visual Analog Scores. Table 6: Physical therapy outcomes. Range of Motion
Increased Knee Cartilage Volume Using Autologous Mesenchymal Stem Cells It should be noted that without biopsy, there is no The pre- and post-procedure MRI analysis demon- way to determine if the change was fibrocartilage or strated an increase in meniscus and cartilage volume true hyaline cartilage. Of interest, this was a "needle that was more than the standard deviation of the 3 out/needle in" procedure with no activity restriction. region of interest measurements. At 3-month follow- Longer term follow-up of this patient will continue. up, modified VAS scores decreased by 95%, from 4 to Obviously, the generalizability of this technique to the 0.38. Range of motion in extension increased from -2 larger population of patients with symptomatic osteo- degrees to +3 degrees with an associated decrease in arthritis and traumatic knee injury is unknown. VAS pain score.
Although no conclusion can be made from one case report, if similar findings are published from pi- onclusion
lot studies and then larger well-designed trials, the This case report shows MRI evidence of increased results may have implications for interventional pain meniscus volume and femoral cartilage volume. While management. For example, while the laboratory ex- there has been evidence from animal models of car- pansion was carried out using specialized lab person- tilage regeneration using MSCs, this is the first case nel, the actual clinical lab was part of an interven- report of an increase in meniscus size in a human sub- tional pain management practice. In addition, the ject (of which we are aware). While the patient's re- clinical procedures used to extract cells via fluoroscopy ported clinical response could have been due to the involved placing a sterile trocar into key bony land- dexamethasone injection provided post transplant marks easily visible via fluoroscopy. In addition, cell procedure, the levels injected (10 ng/mL) were approx- reimplantation was a simple intraarticular knee injec- imately one million times lower than those used clini- tion via fluoroscopy.
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Treatment of Chronic Hepatitis C - September 2016 Update SASL-SSI Expert Opinion Statement (The changes compared to the January 2016 Update are highlighted in yellow) Written by: Jan Fehr, Darius Moradpour, Andri Rauch and Beat Müllhaupt Reviewed and approved by: Philip Bruggmann, Andreas Cerny, Andrea De Gottardi, Markus H. Heim, Beat Helbling, Francesco Negro, Laura Rubbia-Brandt, David Semela and Christian Toso for SASL as well as Hansjakob Furrer, Claude Scheidegger and Pietro Vernazza for SSI.