Mary Beth McCarthy

Platelet-Rich Plasma Differs According to Preparation Method and Human Variability

BACKGROUNDnVarying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma.nnnMETHODSnThree different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated.nnnRESULTSnThe content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed.nnnCONCLUSIONSnSingle-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

An in vitro model for mineralization of human osteoblast-like cells on implant materials.

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

Regioselectivity in the cytochromes P-450: Control by protein constraints and by chemical reactivities

Three alicyclic compounds (D-camphor, adamantanone, adamantane) were found to be hydroxylated by the cytochrome P-450 isoenzymes P-450cam and P-450LM2. With P-450cam as the catalyst only one product was formed from each of the substrates: 5-exohydroxycamphor, 5-hydroxyadamantanone, and 1-adamantanol. With P-450LM2 as the catalyst, two or more isomeric products were formed from each substrate: 3-endo-, 5-exo-, and 5-endo-hydroxycamphor; 4-anti- and 5-hydroxyadamantanone; and 1- and 2- adamantanol. The products from P-450cam hydroxylations were found to be isosteric with one another, suggesting that each of them was attacked at a topologically congruent position within a rigid enzyme-substrate complex. The distribution of products from P-450LM2 hydroxylations, on the other hand, were similar to the distributions expected during solution-phase hydroxylations. Thus, it would appear that the complex which P-450LM2 forms with its substrate allows considerable movement of the substrate molecule, such that most of the hydrogens in the substrate are exposed to the enzymatic hydrogen abstractor. Under these conditions, the distribution of products more nearly reflects the rank order of chemical reactivities of the various hydroxylatable positions, with only a moderate protein-based steric constraint being expressed. These suggestions were also evident in the tightness of binding of the substrates to the two enzymes and in the magnitude of coupling between the substrate binding and the spin-state equilibria. Thus, the product from P-450cam-catalyzed hydroxylation may be predicted by a consideration of the relation of the topology of the prospective substrate to that of D-camphor. The products from P-450LM2-catalyzed hydroxylations, on the other hand, may be approximately predicted from the chemical reactivities of the various abstractable hydrogens in the prospective substrate.

Bone Marrow–Derived Mesenchymal Stem Cells Obtained During Arthroscopic Rotator Cuff Repair Surgery Show Potential for Tendon Cell Differentiation After Treatment With Insulin

PURPOSEnThe purpose of this study was to determine whether a one-time physiologic dose of insulin when compared with the growth factors insulin-like growth factor 1, β-fibroblastic growth factor, and growth differentiation factor 5 is capable of differentiating bone marrow-derived mesenchymal stem cells (MSCs) into tendon.nnnMETHODSnEleven patients undergoing arthroscopic rotator cuff repair consented to undergo aspiration of bone marrow. A dose-response curve was calculated to determine the optimal dose of insulin needed to differentiate MSCs into tendon. After purification of bone marrow in the operating room, MSCs were exposed to either insulin or tendon-inducing growth factors or were left untreated to serve as a control. The potential for MSCs in each of these groups to differentiate into tendon was evaluated with a multistep process that included determination of the genetic upregulation for tendon-specific proteins, confirmation that the levels of these proteins were actually increased, staining of the MSCs with antibodies for these proteins to ensure that they were expressed on the cell surface, and finally, evaluation of cell morphology to verify the MSCs tendon-like appearance.nnnRESULTSnMSCs treated with insulin showed increased gene expression of tendon-specific markers (P < .05), increased content of tendon-specific proteins (P < .05), and increased receptors on the cell surface (P < .05) compared with control cells. Histologic analysis showed a tendon-like appearance compared with the control cells.nnnCONCLUSIONSnBone marrow-derived MSCs treated with a single physiologic dose of insulin differentiated into cells with characteristics consistent with tendon.nnnCLINICAL RELEVANCEnThe potential for MSCs to differentiate into tendon after a 1-time dose of insulin may assist in developing practical biologic options for augmentation of rotator cuff repairs.

Histological and molecular analysis of the biceps tendon long head post-tenotomy

Tendinopathy is a vexing clinical problem as its onset and development is often asymptomatic and unrecognized until tendon rupture. While extensively studied in the rotator cuff, Achilles, and patellar tendons, no study to date has examined the histological and molecular characteristics of the tendinopathic biceps long‐head (LHB). The anatomy of the LHB is unique in that it comprises intra‐ and extra‐articular portions, each exposed to differing loading patterns. Eleven LHBs post‐tenotomy were sectioned, fixed in formalin, and stained (H&E; Alcian Blue), and gross structural organization of collagen measured using polarized light microscopy. Protein expression of intra‐ and extra‐articular portions of the tenotomized biceps for IGF‐I, collagen III, and MMP‐1, ‐2, ‐3, and ‐13 was determined with Western blot analyses. The intra‐articular LHB exhibited significantly greater histological evidence of tendinopathy inclusive of increased proteoglycan (pu2009<u20090.05) and decreased organization as measured by polarized light microscopy (pu2009<u20090.01). The intra‐articular LHB also had significantly increased expression of collagen type III (pu2009<u20090.01) and of MMP‐1 and 3 (pu2009<u20090.01, pu2009<u20090.05 respectively). No significant differences were found for IGF‐I or for MMP‐2 and ‐13. The intra‐articular LHB exhibited histological characteristics of tendinopathy. Protein expression of the intra‐articular LHB did not universally display signs of tendinopathy in comparison to the extra‐articular portion of the tendon.

Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture

Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five‐step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma‐derived cell line, SaOs‐2. No significant differences were found in DNA synthesis, assayed by [3H]‐thymidine incorporation at 1 or 2 weeks for SaOs‐2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (pu2009= 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (pu2009= 0.016) and decreased mineralization in SaOs‐2 (pu2009= 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT‐induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs‐2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma‐derived cell line.

Biologics in shoulder surgery: The role of adult mesenchymal stem cells in tendon repair

Successful treatment of chronic and large rotator cuff tears presents a challenge to the orthopaedic surgeon. There are times when no optimal reparative or reconstructive option exists, as in the case of massive or chronic rotator cuff tears, and “salvage” operations are the only reasonable surgical options. More recently is the emerging paradigm of utilizing adult mesenchymal stem cells (MSCs) and bioscaffolds to replace diseased or damaged tendon, cartilage, and bone. Over the last decade, there has been an explosion of literature defining the properties and potential role of adult MSC in the treatment of orthopaedic problems. Early evidence indicates that adult MSCs are capable of regenerating diseased tissue. Moreover, MSCs are easily obtainable and amplifiable with current tissue engineering technology, and they appear to be immunologically tolerated. Isolation and culture expansion of human MSCs will likely provide novel approaches to the treatment of many complex clinical problems. The present account reviews the current knowledge of mature MSCs as it relates to tendon healing. In addition, we present preliminary results of our investigations.

Biologic enhancement of a common arthroscopic suture.

PURPOSEnThe purpose of this study was to investigate the in vitro effects of an arginine-glycine-aspartic acid (RGD) coating on a high-strength nonabsorbable polyester/polyethylene (PE/PEE) suture material commonly used in orthopaedic procedures.nnnMETHODSnHuman bone and tendon specimens were isolated and cultured. The cells were then plated in known densities in the presence of RGD-coated and uncoated PE/PEE suture and allowed to adhere for predetermined time periods. The RGD-coated and uncoated control sutures were then removed and assayed for cell osteoblast and tenocyte adhesion and proliferation.nnnRESULTSnThe RGD-modified suture showed a statistically significant increase in both adhesion and proliferation of human tenocytes when compared with uncoated controls (P < .05).nnnCONCLUSIONSnThe RGD peptide sequence can be effectively coupled with commercially available PE/PEE suture. RGD-coated suture is able to stimulate the adhesion and proliferation of human tenocyte cells in vitro, as well as withstand standard sterilization and storage conditions. Furthermore, the acid hydrolysis process did not affect the strength of the suture material.nnnCLINICAL RELEVANCEnRGD-modified suture materials have the potential to create favorable biologic responses when used in common orthopaedic procedures.

Extracellular Matrix of Current Biological Scaffolds Promotes the Differentiation Potential of Mesenchymal Stem Cells

PURPOSEnThe purpose of this study was to quantitatively assess the ability of bone marrow-derived mesenchymal stem cells (bMSC) to differentiate toward bone, fat, cartilage, and tendon lineages when grown on commercially available scaffolds compared with control and native tendon tissue.nnnMETHODSnBMSCs were cultured and analyzed by fluorescent automated cells sorting for surface markers CD73, -90, and -105. BMSCs were grown on rotator cuff tendon (RCT), decellularized human dermis patch (DDP), bilayer collagen matrix, and fibrin matrix (FM) to test their differentiation potential using quantitative polymerase chain reaction and establish markers for osteogenic, adipogenic, chondrogenic, and tenogenic lineages. Immunocytochemical testing was used to determine the specific proteins present on the scaffolds.nnnRESULTSnAlkaline phosphatase and osteocalcin gene expression was significantly higher on RCT (P < .001) and collagen scaffold (CS) (Pxa0<xa0.001) compared with DDP and FM scaffolds (P < .001, P < .001). When differentiated toward a cartilage lineage, bMSCs grown on CS had significantly more type II collagen and aggrecan compared with DDP (P < .001, P < .001), FM (Pxa0< .001, P < .001), and RCT (P < .001, P < .001). Differentiated bMSCs grown on the CS had a significant increase in PPARγ and FABP4 gene expression compared with bMSCs grown on all other scaffolds (all P < .001). The differentiation of bMSCs into tendon on CSs had significantly more tenacin C, decorin, and type III collagen gene expression when compared with RCT, DDP, and FM (all P < .001). Decorin gene expression in the control undifferentiated CS wasxa0alsoxa0significantly increased, suggesting that the matrix alone may promote a tenogenic lineage (Pxa0= .637).nnnCONCLUSIONSnDifferences in the extracellular matrix composition of scaffolds significantly impact their potential to promote differentiation of bMSCs. Comparing the native RCT to the tested scaffolds showed that a high content of type I and III collagen significantly increased the potential of bMSCs to differentiate toward bone, tendon, fat, and cartilage lineages.nnnCLINICAL RELEVANCEnThis inxa0vitro study shows the differences between commercially available scaffolds for rotator cuffxa0repairs. Therefore, these results support clinical use depending on the surgical intention and the potential of bMSCs to differentiate into bone, tendon, cartilage, and fatxa0tissue.

Bone Marrow-Derived Mesenchymal Stromal Cells Enhanced by Platelet-Rich Plasma Maintain Adhesion to Scaffolds in Arthroscopic Simulation

PURPOSEnTo assess the response of bone marrow-derived mesenchymal stromal cells (bMSCs) enhanced by platelet-rich plasma (PRP) in the setting of a normal human tendon (NHT), a demineralized bone matrix (DBM), and a fibrin scaffold (FS) with simulated arthroscopic mechanical washout stress.nnnMETHODSnBone marrow was aspirated from the humeral head and concentrated. BMSCs were counted, plated, and grown to confluence. Cells were seeded onto 3 different scaffolds: (1) NHT, (2) DBM, and (3) FS. Each scaffold was treated with a combination of (+)/(-) PRP and (+)/(-) arthroscopic washout simulation. A period of 60xa0minutes was allotted before arthroscopic washout. Adhesion, proliferation, and differentiation assays were performed to assess cellular activity in each condition.nnnRESULTSnSignificant differences were seen in mesenchymal stromal cell adhesion, proliferation, and differentiation among the scaffolds. DBM and FS showed superior results to NHT for cell adhesion, proliferation, and differentiation. PRP significantly enhanced cellular adhesion, proliferation, and differentiation. Arthroscopic simulation did not significantly decrease bMSC adhesion.nnnCONCLUSIONSnWe found that the type of scaffold impacts bMSCs behavior. Both scaffolds (DBM and FS) were superior to NHT. The use of an arthroscopic simulator did not significantly decrease the adhesion of bMSCs to the scaffolds nor did it decrease their biologic differentiation potential. In addition, PRP enhanced cellular adhesion, proliferation, and differentiation.nnnCLINICAL RELEVANCEnImproved healing after tendon repair can lead to better clinical outcomes. BMSCs are attractive for enhancing healing given their accessibility and regenerative potential. Application of bMSCs using scaffolds as cell carriers relies on arthroscopic feasibility.