The document discusses the historical context and applications of orthobiology, particularly focusing on embryonic stem cells and biologic substances used for healing musculoskeletal conditions. It examines various biological treatments, including platelet-rich plasma (PRP) and bone morphogenic proteins (BMPs), emphasizing their roles in bone healing, regeneration, and the regulation of differentiation in stem cells. Clinical applications, mechanisms of action, and issues with traditional treatments for conditions like arthritis are also highlighted, presenting PRP as a promising alternative for managing tissue injuries.

1. ORTHOBIOLOGY
;

2. ONCE UPON A TIME...
Wednesday, 4 June 2
3000 BC : As perMahabharata,Adi Parva,
chapter 114,shloka17, theKauravawerecreatedby
splittingthe singleembryointo101partsandgrowing
eachpartina separate kund or container. It is
believedthatKaurva’s birthmentionedhereisfirst
reportedmentionofwhatcould bepossibleuseof
embryonic stem cell.

3. Orthobiologics:
• Ortho: to make straight or right
• The use of biologic substances to prompt, stimulate or support a “healing
event” within the body
• The use of biologic substances to promote healing or reduce pain
• The use of platelets and stem cells in treatment and management of
musculoskeletal conditions

4. ◦ Within orthopedics, focal chondral lesions, osteo- arthritis (OA), fracture
healing, and soft tissue repair (tendon to tendon interface, tendon to bone
interface, meniscus, muscle, and ligaments) are fertile fields for biological
therapies.
◦ Biological and surgical treatments have been proposed to treat these
pathologies.
Biological treatments include
◦ platelet-rich plasma (PRP),
◦ bone marrow aspirate concentrate (BMAC),
◦ and cell-based therapies.
◦ Surgical interventions include marrow stimulation procedures and stem cells
implanted in matrix

5. BMP’S IN ORTHOPEDICS
◦ Bone Morphogenic Proteins (BMPs) are a group of molecules which induce
mesenchymal stem cells to differentiate into bone forming cell lines that form
new bone.
◦ They are a group of noncollagenous glycoproteins that mostly belong to the
transforming growth factor-beta (TGF-b) superfamily.
◦ BMPs have been widely used in the field of orthopaedics since the last two
decades.
◦ BMPs are a group of osteoinductive proteins that are usually extracted from
bone matrix which are responsible for skeletal regeneration and bone healing.

6. ◦ The primary functions of BMPs are to activate inactive mesenchymal
stem cells and to differentiate them into osteoblast, chondroblasts and
fibroblasts.
◦ Apart from the above functions, BMPs are also involved in other
physiological and pathological processes such as inflammatory response,
bone formation and resorption, growth signalling pathways,
oncogenesis and immune response.
◦ The safety and efficacy of BMP as bone graft substitute are influenced by
its purity, local effects, systemic effects, immunogenicity and
biocompatibility.

7. ◦ Bone ossification occurs through both intramembranous ossification and
endochondral ossification
◦ Both cellular events of intramembranous ossification and endochondral
ossification involve MSCs which may be bone marrow derived or
periosteum derived.
◦ These primitive MSCs are pluripotent progenitors that can differentiate
into both osteoblasts and chondroblasts.
◦ This differentiation is regulated by molecules such as BMPs and
Fibroblast growth factor (FGF).

8. Classification of BMPs
• Till date more than 20 BMPs have been identified in vertebrates.
• Based on structural homology, BMPs can be further classified into severa
subgroups which includes BMP-2/- group, BMP-5/-6/-7/-8 group, BMP-9/ 10
group and BMP-12/-13/-14 group.

9. Functions of BMPs
◦ The actions and functions of BMPs depend on various factors which
are:
◦ 1. Type of target cell.
◦ 2. Maturation phase of target cells.
◦ 3. Local concentration of BMPs.
◦ 4. Other biological signals.

10. Functions of BMPs
◦ BMPs are mitogens (growth factors) that stimulate the production and
multiplication of morphogens (differentiation factors) that transform
connective tissue cells into osteoprogenitor cells.
◦ They play a pivotal role in embryonic development through specification of
positional information in the embryo.
◦ They regulate growth, differentiation, chemotaxis and apoptosis of cells such
as epithelial, mesenchymal, haematopoietic and neuronal cells.
◦ Apart from inducing bone formation through intramembranous or
endochondral ossification, they also induce osteoclasts leading to bone
resorption.

11. Carriers of BMPs
• BMP is a water soluble molecule
which diffuses into the body fluid
easily.
• When administered alone, most of the
protein molecule is lost rapidly due to
diffusion and irrigation.
• To negate these effects and have a
prolonged localized effect at target
site, BMPs are administered in a
carrier.
• Type I collagen is the most preferred
carrier used for transport of BMPs.

12. Clinical Applications in Orthopaedics
◦ 1. BMP as an alternative to autograft for non-unions Long Bones –
◦ Currently, US Food and Drug Administration (FDA) approval of BMP use
in long bones is limited to tibia non-unions as alternative to autograft.
◦ Pseudoarthrosis of tibia remains to be the most frequent site of long bone
non-union due to its poor muscular coverage, high incidence of open
injuries and its anatomical peculiarity.
◦ Friedlaender et al initially reported the efficacy of BMP-7 in the treatment
of tibia non-unions.
◦ It was concluded that BMP-7 provided comparable results when compared
with bone autograft, without donor site morbidity.

13. Scaphoid
◦ Scaphoid is very commonly affected with non-union due to its precarious
vascular supply.
◦ Patients treated with autograft and BMP-7 healed successfully within 3
months
◦ However, all the evidences on scaphoid non-unions were poor.
◦ SPINE
◦ By far, spine is the most common site associated with BMP application.
◦ US FDA has approved the usage of rhBMP-2 in anterior lumbar interbody
fusions.
◦ However recent studies have shown that BMP use has been extended to
fusions performed with other approaches as well.

14. ◦ BMP as an alternative to autograft for fusion Foot and ankle –
◦ Few authors have used BMPs to improve the rates of union in ankle
arthrodesis for patients with comorbidities affecting bony union.
◦ Initially use of BMP in complex foot and ankle arthrodesis seem to be a
safe option considering its high failure rates.
◦ However, there is no randomized control trial available till date to give a
suggestable conclusion.

15. Platelets
◦ Platelets develop from megakaryocytes in the marrow.
◦ Each megakaryocyte can produce 5000- 10000 platelets. Approximately 2/3rd
of the platelets circulate in the blood and 1/3rd are stored in the spleen.
◦ An average healthy adult can produce 1011 platelets per day.
◦ The normal platelet count is (150– 400) × 103 per microliter of blood.
◦ The diameter of a mature platelet is 2-3 5μ.
◦ Platelets usually remain alive for 5-11days.
◦ Old platelets are destroyed by phagocytosis in the spleen and liver by Kupffer
cells

16. +
What is the response to platelets
• Proliferation of Mesenchymal
stem cells
• Proliferation of fibroblasts
• Production of Type 1 Collagen
• Platelets itself release Growth factors

17. Healing pathway

19. PLATELET RICH PLASMA
◦ By definition, Volume of plasma fraction of autologous blood having a platelet
concentration above baseline.
◦ To be labeled as PRP, a platelet count of 3-5 times of the baseline should be
present in the platelet concentrate(many studies like Max et al mention the
platelet count 1 million as standard valve for PRP).
◦ Platelets begin secreting these proteins within 10 minutes of activation.
After initial release of growth factors, the platelets synthesize and secrete
additional factors for the remaining days of their life span.

20. ◦ In humans, the normal platelet count in whole blood ranges from
approximately 150,000 to 350,000/μL ,
◦ whereas platelet- rich plasma is often defined as at least 1,000,000
platelet/ μL suspended in plasma.
◦ In order to ensure that the platelets are suspended and do not form a
clot, PRP must be made from anticoagulated blood.
◦ The logic behind PRP is that platelets are the first to arrive at the
site of tissue injury, and thus have the potential to release growth
factors that play a critical role in mediating healing

21. History
◦ The term PRP first used by KINGSLEY
◦ In 1970, MATRAR used fibrin glue in rats
◦ 1998 –PRP in regenerative medicine for bone healing
◦ 2001- 2nd generation platelet concentration identified

22. ◦ Physicians report that the demand for PRP has soared after pro golfer
Tiger Woods received injections to accelerate healing after knee
surgery.”

23. What is PRP
◦ Platelet Rich Plasma
◦ Utilizing growth factor (GF) content of platelets to aide in healing of
musculoskeletal tissue
◦ Predominately tendons, ligaments and muscles
◦ High concentration of GF deposited locally in the area
of an injury
◦ Anabolic effect enhances and supports healing

25. +
What does it lead to??
• Chemotaxis
• Directional movement in response to a chemical stimulus
• Stems cells are attracted to the growth factors and migrate
into the area
• Cell proliferation
• Significant increase in cellular reproduction activity
• Possibly even a systemic effect
• Performance enhancing?

27. PRP – MECHANISM OF ACTION
1st Mechanism:
• Delivery of Growth Factors
2nd Mechanism :
• “Up regulation” of local GF in tendons
• TGF-Beta1 for 1st week post prp,
• Increased IGF-1 for 4 weeks tenocytes post prp

28. 3rd mechanism:
◦ PRP Stimulates HA Release
• PRGF significantly enhanced HA secretion compared with platelet-
poor preparations
4th mechanism :
◦ Chemo-attractant for Stem Cells
• Prompt Migration and Proliferation

29. Platelets
◦ Although platelets play a key role in haemostasis, they are central in mediating
the anabolic effects of PRP by virtue of releasing growth factors stored in their
alpha granules.
◦ During the initial phases of wound repair, activated platelets attract and foster
cell migration into the wound by aggregating and forming a fibrin matrix.
◦ This matrix then serves as a tissuescaffold for sustained release of platelet
growth factors and cytokines, which stimulate cell recruitment, differentiation,
and communication

30. 2. Leukocytes
◦ Leukocytes are essential mediators of the inflammatory response, host defence
against infectious agents, and wound healing.
◦ Neutrophils are involved in the inflammation phase of wound healing.
◦ Monocytes and macrophages facilitate tissue repair by debriding and
phagocytising damaged tissue and debris.
◦ Similar to platelets, macrophages also secrete growth factors that are important
in tissue repair and have been shown to contribute to subchondral bone
regeneration.

31. 3. Red blood cells
◦ Red blood cells (RBC) content is typically reduced or absent in PRP
because of the centrifugation process.
◦ Destructive process is thought to occur in human synoviocytes treated
with RBC concentrates, leading to significantly greater cell death and
cartilage degradation.

32. Classification of PRP
Dohan et al in the year 2009 classified PRP based on presence of
leukocytes and density of fibrin network

33. Mishra et al in the year 201 proposed a classification system which
is based on platelet count, activation and leukocytes for clinical
application in Sports medicine

34. +
Advantages of having leucocytes??
Pure PRP has an advantage over traditional PRP in that it
eliminates red blood cells (RBCs) and neutrophils.
RBCs, have no therapeutic effect and more painful while
injecting.
Neutrophils, a type of white blood cell, have inflammatory
components which may increase pain and inflammation
post-treatment.

35. ◦ Equipment required for processing 40ml venous
blood (figure1)
1. Centrifuge (Remi R-4C), 4000rpm with countdown timer fixed angle
rotor, capacity 4x50 ml
2. First centrifugation (soft spin) : 2x20cc syringe, 6ml ACD-A (Acid citrate
dextrose), 2xBD insulin syringe
3. Second centrifugation (hard spin): 20cc syringe, 3-way cannula, BD
insulin syringe
4. PRP extraction: 10cc syringe, 3-way cannula.

36. STEP 1
◦ Three ml ACD-A was taken in 20ml syringe and 17 ml blood drawn from
the ante cubital vein. Blood (0.3 ml) was sent for initial platelet count.
◦ Nozzle of the syringe was blocked firmly with the cap of the insulin
syringe. With a heavy scissors flanges on the either side of the syringe
barrel were cut.
◦ The piston was cut leaving one inch projecting out of the barrel.
◦ The two syringes were placed in centrifugation tubes opposite to each
other with the tip pointing upwards.
◦ This will help balance the centrifuge and minimize vibration.
◦ The first spin was set at 1500rpm for 15mins without brake (Soft spin).

38. STEP II
◦ After soft spin blood in the syringe was separated into three layers.
◦ The bottom layer comprises of RBC, the middle layer leucocytes and the top
layer, plasma with platelets in suspension.
◦ The serum was drawn into the second 20cc syringe through the 3-way cannula.
Care was taken to avoid aspiration of the buffy coat and RBC.
◦ The combined volume of plasma from both syringes ranged between 16-18ml.
◦ The syringe was again capped and modified to fit the centrifugation tube.
◦ The second spin was set at 3500rpm for 7minutes (Hard spin).

40. STEP III
◦ At the end of second spin the
syringe contains Platelet Poor
Plasma (PPP) on top and platelets
along with scanty WBC at the
bottom in the form of a pellet.
◦ The supernatant plasma is drawn
and discarded leaving about four ml
at the bottom which is reconstituted
to form PRP fraction. This is sent
for culture and counts.

41. RESULTS

43. PRF
◦ Under strict aseptic conditions, ten to twenty millilitres of patient's
blood was taken and centrifuged at 3000 rpm for 15 min.
◦ PRFM was separated from red corpuscles at the base using a sterile
forceps and scissor, preserving a small RBC layer measuring around
one mm in length, which was transferred onto a sterile gauze

45. ◦ Middle membrane so obtained was compressed between two gauze pieces
gently and applied on a healthy wound followed by application of a
secondary non-absorbable dressing.
◦ The secondary dressing of the patient and the dried PRFM was removed
from the wound bed after a minimum of five days.
◦ The same procedure was repeated every week for six weeks.

46. +
Where can it be used??
 Everything and anything!
 Internet marketing for host of ailments
 Snake oil of today
“Cures Rheumatism, Sports injuries, Grows hair,
Gets rid of wrinkles, etc., etc.”

47. +
 Meniscus repair
 Tendonitis (office based injection)
 Arthritis
 Ligament surgery
 Tendon repairs
 Acute injury – Debatable!

48. +
 Epicondylitis, Achillodynia
 Runners / Jumpers Knee
 Plantar fascitis
 Osteoarthritis (Grad I-III)
 Muscle- and Ligament Lesions - Ultrasound-Guided
Injections
 Peri – Post operatively
- Achilles Tendon Repair
- HTO, Microfracturing
- Meniscal Suture, RC Repair etc

49.  Analgesic?
 Potential primary analgesic effect
 Some human studies state decreased post-op pain
levels
 Stimulation of thrombin receptors (ie, PAR-1) shown
to increase pain threshold in laboratory animals
through opioid pathways and haemostasis
 Antimicrobial
 Against Staphylococcus aureus (Sutter 2012)

50. Arthritis
What happens in an
arthritic joint?

51. • Cartilage wears out
• chondrocytes unhealthy/poor
quality matrix - breaksdown
• Poor lubrication - hyaluronic
acid/lubricin (SZP -Proteoglycan
4)
• increased friction - wears out
Inflammation - pain

52. Arthritic Joint - The “BAD” Molecules
• MMP’s - matrix metalloproteinases - collagenases, gelatinases,
stromelysins - degrade collagen, proteoglycans, elastin, etc.
• IL-1, TNF - interleukins - inflammatory - enhance MMP’s
• ADAMTS - disintegrins - prevent platelet function
• CATABOLIC - BREAKDOWN

53. What About “GOOD” Molecules?
• TGF - tissue growth factor
• TIMP-1&2 - tissue inhibitor of metalloproteinases
• IRAP - interleukin receptor antagonist protein
• A2M - alpha 2 macroglobulin (useless by itself
• - so drug companies barking up the wrong tree)
• Block the BAD molecules -
• ANTI-CATABOLIC (ANABOLIC)

54. PRP (Platelet Rich Plasma)
Reduces “BAD” Molecules
Increases “GOOD” Molecules

55. What About “Traditional” Treatments?
Are there problems?
NSAIDS
Corticosteroid
Surgery
(Physical Therapy ALWAYS GOOD!)

56. NSAIDS
• Commonly taken
• Multitude of side-effect Very significant
problems
• ulcers, bleeding, heart attack, stroke, kidney and liver
failure
• Inhibit chondrocytes and stem cells

57. Corticosteroids Injections
• Commonly performed
• Temporarily reduce inflammation
• Inhibit healing
• Cause damage to cartilage and soft tissue structures

58. PRP Intra-articular Pathways
• Decreases BAD molecules - MMP, IL - reduces
CATABOLIC PROCESSES
• Increases GOOD molecules - TGF, TIMP, IRAP,
A2M
• REBALANCES the micro- environment of the joint -
back to favoring the normal
healing status

59. • Produced by synovial cells
• Important for lubrication/viscosity of joint fluid
• Hyaluronic Acid - Hyaluronan
• Viscosupplementation -Synvisc, Orthovisc - from rooster
combs
• HA alone not enough to protect the joint
PRP Stimulates Hyaluronic Acid
Production

60. +
Tennis elbow
• Common Disorder
• Repetitive activities
– Most common cause
– May occur from injury (acutely)
• PRP Application Technique
– Inject 2-3 cc of PRP into the ECRB--Peppering
technique
– Average Dose 3.3 million platelets per patient

61. Lateral Epicondylitis - PRP
• PRP provides excellent and
superior results
• Results are longer lasting
• Particularly better than
corticosteroid injection
Am J Sports Med 2006, 2014, J Clin Diag
Res 2015, J Hand Microsurg
2015

62. Compared to Surgery:
PRP - showed 83% improvement
Surgery - showed 46% improvement
PRP improved 2X as much as
surgery
SO WHY ARE WE STILL DOING
SURGERY FOR THIS PROBLEM?
J Orthopaedics March 2016

63. Planar Fasciitis
PRP
• more effective and durable than
corticosteroid
• 3X (300%) the functionality and
improvement of pain
• Actually heal structures - rather than mask Sx (Foot Ankle Int, April 2014)

65. Shoulder Impingement
• Rotator cuff tendons weaken, develop tears,
degenerative changes
• Poor blood supply Unable to heal
itself
• Gets stuck in early phase of repair Chronic
changes, inflammation, pain Poor healing
environment

66. Shoulder
Impingement
NSAIDS –
minimal benefit, inhibit healing
Corticosteroids -
if used - no longer see healing response - Br J Sports
Med 2014
kill rotator cuff cells -Bone Joint Res 2014

67. Shoulder Impingement - PRP
• PRP more effective than corticosteroid
• in pain relief (30% better)
• in ROM improvement (3X more
improvement)
• 16X less likely to undergo surgery
(3/100 vs 48/100)
O’Donnell, et al. AAOS 2013

68. +
TendoAchilles

70. +
Post Injection Therapy
• Rest (sling/crutches) for a few days to a week
• NO NSAID’S
• Slow stretching program
• No high loading activities until
• No rest pain
• Minimal tenderness

71. +
COMPLICATIONS
 No adverse complications have been reported
 Symptoms may be worsened for few days because of induced
inflammation
 Infection
 Persisting symptoms

72. +
Limitations
• Efficacy
• Lack of clinical data
• Number of studies being done
• Indications are being developed
• Uniformity of prep
• PRP classification
• Insurance recognition

75. Stem Cell
• An undifferentiated progenitor/primitive cell
• A: capable of renewing itself through cell division, sometimes after long
periods of inactivity
• B: Under certain physiologic or experimental conditions, it can be
induced to become a tissue- or organ-specific cell with special
functions

76. STEM CELL RESEARCH TIMELINE

77. STEM CELL CHARACTERISTICS
Wednesday, 4 June 14 77
Undifferentiated cells with the capacity for unlimited or
prolonged self-renewal that can give rise to
differentiated cells.
Self renewing for long periods.
Unipotent /pluripotent /multipotent e.g. Intestinal stem
cells produce one or more of the 4 classes of mature
differentiated cells (absorptive, goblet, Paneth and
enteroendocrine cells)

78. Sources of stem cells
◦ Adult Tissue: Hematopoeitic SC,
Mesenchymal SC, Neural, muscle
derived, Pancreatic, Hepatic,
Epithelial, Dental.
◦ Fetal tissue
◦ Cord blood
◦ Embryos

82. BMAC
◦ In adult skeleton there are two types of bone marrow which are red marrow
and yellow marrow.
◦ Yellow marrow is filled with adipose tissue and is inactive by function. Red
marrow possesses hematopoietic cells and mesenchymal stem cells.
◦ Mesenchymal stem cells (MSC) have the capability to differentiate into cell
lines like cartilage, bone, tendon, muscle and nerves.
◦ Benefits of BMAc are achieved either by proliferation and differentiation into
variety of cell lineages or by elution of growth factors and cytokines to hasten
the process of healing in tissues with attenuated healing potential.

83. Components of BMAC
◦ In normal bone marrow aspirate (BMA),common components present are
erythrocytes(22-28%), neutrophils (32-37%),lymphocytes(13%), eosinophils
(2.2%), blast cells(1.4%), monocytes (1.3), basophils(0.1%) and megakaryocytes
in variable percentages.
◦ Apart from the above components, BMA contains mesenchymal stem cells and
various growth factors.
◦ An adult bone marrow has about 0.001% to 0.01% of mesenchymal stem cells in
7-30 cells per million nucleated cells.
◦ Growth factors like PDGF, TGF-â, BMP2and7 and IL-1RA are also present in the
BMA in variable concentrations

84. BONE MARROW CONCENTRATE
Wednesday, 4 June 14 84
Enhances levels of Bone Marrow-BM
Mesenchymal Stem MS) Cells
BM-MS cells decrease with age
Concentrating mimics the cell load and healing
potential of young patients
4-5x progenitor cell increase above baseline

85. ◦ Main aim of bone marrow concentration is to increase the cell count of
mesenchymal stem cells.
◦ The concentrations of MSC in bone marrow alone is relatively low, hence
the aspirate is concentrated by centrifugation in order to increase the
percentage of MSCs.
◦ Cell count in bone marrow aspirate is 612±134 per cm3 compared to bone
marrow aspirate concentrate is 2579±1121 per cm3 which is a fivefold
increase in the number of mono nuclear cells

88. Sources of BMAC
◦ Commonest source of bone marrow aspiration is from iliac crest, tibia
or calcaneum.
◦ Among all of the sources,iliac crest yields highest concentration of
mesenchymal stem cells.
◦ Graft is harvested bysector rule in iliac crest, which roughly divides the
entire iliac crest into six zones.
◦ Of these six zones the posteriorcrest is preferred site as it contains
higher concentration of MSCs (higher by 1.6 times compared to
anterior iliac crest).

89. ◦ The patient can be positioned either supine or prone. In the supine
position, BMAC is harvested from the anterior superior iliac spine or
the iliac crest. In the prone position, BMAC is harvested from the
posterior superior iliac crest region.
◦ Monitored anesthesia (conscious sedation), local anesthesia, or
general anesthesia can be used for this BMAC harvest procedure, we
prefer positioning the patient in the prone position with light
sedation monitored by an anesthesiologist for access to the posterior
superior iliac crest.
◦ After palpation of the bony landmarks, the procedural site is
sterilely prepared and widely draped to ensure an adequate field.

90. • The BMAC aspiration kit is opened and then the battery-powered BMAC
aspiration device is sterilely drape
• A bone marrow aspiration kit (MarrowStim; Biomet Biologics, Warsaw, IN) is
used for bone marrow aspiration
• First the bony landmarks of the posterior iliac crest and sacroiliac joint are
palpated. The skin is then injected down to and including the periosteum with 1%
lidocaine without epinephrine.

91. • Then, the bone marrow aspiration
trochar and needle are percutaneously
inserted through the skin and
subcutaneous tissues until it reaches
the posterior iliac crest.
• Then, manual pressure is used to
position the bone marrow aspiration
trochar against the dense cortical bone,
attempting to center it over the middle
of the posterior crest cortical walls.
• The trajectory of the needle should be
parallel to the iliac crest, or
perpendicular to the ASIS or PSIS,
depending on the harvest site used

92. • A battery-powered power instrument is then used to drill the trochar and
needle into the medullary cavity of the posterior iliac crest
• After the trochar is inserted into the posterior iliac crest but prior to
aspiration, 1 mL of heparin (1,000 U/mL) should be preloaded into the
syringe.
• Preloading the needles avoids clot formation and coagulation, which can
diminish the ultimate yield from the aspiration.
• Approximately 60 mL of bone marrow is aspirated, which requires the use of
two 30-mL syringes.

93. Processing of the Bone Marrow
Aspirate
◦ The bone marrow aspirate (BMA) sample must be processed after it is
harvested. BMA is filtered through a 200-μm-mesh filter into 50-mL
conical tubes.
◦ then, 1 to 1.5 mL of the filtered BMA is pipetted into a 2-mL
microcentrifuge tube for hemanalysis and the sample complete blood
count with differential is automatically recorded.
◦ Subsequently, 60 to 90 mL of BMA is transferred into 2 × 50-mL conical
tubes, and initially centrifuged at 2,400 rpm for 10 minutes.

94. ◦ After completion of this process, the buffy coat layer and
platelet-poor plasma layer are extracted from the conical tube
and discarded.
◦ The red blood cell layers are combined into 1 × 50-mL conical
tube for second centrifugation (3,400 rpm for 6 minutes).
◦ Finally, the BMAC/white cell pellet is resuspended in platelet
poor plasma, hemanalysis is performed and complete blood
count with differential is recorded (including monocyte count) to
assess the final product to inject

95. ◦ After density gradient centrifugation to remove red blood cells,
granulocytes, immature myeloid precursors, and platelets, progenitor cells
account for a small population within the bone marrow (0.001% to
0.01%).
◦ However, a high concentration of growth factors, including platelet-
derived growth factor, transforming growth factor-β, and bone
morphogenetic proteins 2 and 7, which are reported to have anabolic and
anti-inflammatory effects are present in BMAC.
◦ Of note, it has been reported that BMAC has a considerable concentration
of interleukin-1 receptor antagonist (IL-1RA).
◦ This molecule inhibits IL-1 catabolism and therefore may be responsible
for the beneficial symptomatic pain relief with this biologic approach.

96. BMAC USES
Wednesday, 4 June 14 96

97. BMAC IN NON UNIONS
Wednesday, 4 June 14 97

98. BMAC IN SPINAL FUSION
Wednesday, 4 June 14 98

99. BMAC IN AVN
Wednesday, 4 June 14 99

100. BMAC FOR CARTILAGE REPAIR
Wednesday, 4 June 14 100